US5834306A - Tissue specific hypoxia regulated therapeutic constructs - Google Patents

Tissue specific hypoxia regulated therapeutic constructs Download PDF

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US5834306A
US5834306A US08/365,486 US36548694A US5834306A US 5834306 A US5834306 A US 5834306A US 36548694 A US36548694 A US 36548694A US 5834306 A US5834306 A US 5834306A
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Keith A. Webster
Nanette H. Bishopric
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SRI International Inc
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Priority to CA002208599A priority patent/CA2208599A1/en
Priority to PCT/IB1995/000996 priority patent/WO1996020276A1/en
Priority to EP95935540A priority patent/EP0799308A1/en
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Definitions

  • This invention relates to chimeric genes (e.g., carried on expression vectors) containing therapeutic genes whose expression is under the control of tissue specific and hypoxia response enhancer elements.
  • myocardial infarction is due in large part to tissue damage caused by ischemia and/or ischemia followed by reperfusion.
  • the invention includes a method for reducing ischemic injury to a cell exposed to hypoxic conditions.
  • the method includes introducing into the cell a chimeric gene containing a hypoxia response element, a therapeutic gene, and a tissue-specific promoter operably linked to the therapeutic gene to control transcription of the therapeutic gene in the cell, where the element is effective to modulate expression of the therapeutic gene. Exposing the cell to hypoxic conditions enhances expression of the gene and expression of the gene is effective in reducing ischemic injury to the cell.
  • the method may be applied to, for example, cardiac cells using a cardiac-specific promoter, kidney cells using a kidney-specific promoter, brain cells using a brain-specific promoter, and vascular endothelium cells using a vascular endothelium-specific promoter.
  • the hypoxia response element may be selected from, for example, the erythropoietin HRE element (HREE1), muscle pyruvate kinase (PKM) HRE element, ⁇ -enolase (enolase 3; ENO3) HRE element and endothelin-1 (ET-1) HRE element.
  • the therapeutic gene may be selected from, for example, nitric oxide synthase (NOS), B-cell leukemia/lymphoma 2 (bcl-2), superoxide dismutase (SOD) and catalase.
  • the invention includes a chimeric gene, containing a hypoxia response element, a tissue-specific promoter heterologous to the element, and a therapeutic gene.
  • the promoter is operably linked to the therapeutic gene and the element is effective to modulate expression of the therapeutic gene.
  • the method may be used with a variety of cell types and corresponding promoters, for example, as identified above.
  • Suitable cardiac-specific promoters include the ⁇ -MHC 5 .5 promoter, ⁇ -MHC 86 promoter, and human cardiac actin promoter.
  • Suitable kidney-specific promoters include the renin promoter.
  • Suitable brain-specific promoters include the aldolase C promoter and the tyrosine hydroxylase promoter.
  • Suitable vascular endothelium-specific promoters include the Et-1 promoter and vonWillebrand factor promoter.
  • Hypoxia response enhancer element useful with the method include HREE1, PKM HRE element, ENO3 HRE element and ET-1 HRE element.
  • Therapeutic genes useful with the method include NOS, Bcl-2, SOD and catalase.
  • the chimeric gene may also be contained in an expression vector.
  • the expression vector may be a plasmid, adenovirus vector, retrovirus vector, or the like.
  • the invention includes a chimeric gene which contains a hypoxia response element, a tissue-specific promoter heterologous to the element, and a deleterious gene.
  • the promoter is operably linked to the deleterious gene, and the element is effective to modulate expression of the deleterious gene.
  • Suitable promoters include tumor-specific promoters, such as alpha fetoprotein (AFP) promoter.
  • AFP alpha fetoprotein
  • Suitable hypoxia response elements are as articulated above.
  • Deleterious genes useful in this aspect include a viral thymidine kinase gene (tk), such as the herpes simplex virus (HSV) tk.
  • the invention includes a method of causing injury to a cell exposed to hypoxic conditions.
  • the method includes introducing into the cell a vector containing a hypoxia response element, a deleterious gene, and a tissue-specific promoter operably linked to the gene and capable of controlling transcription of the gene in the cell. Exposing the cell to hypoxic conditions enhances expression of the gene, and expression of the gene is effective to cause injury to the cell.
  • Promoters useful with this method include tumor-specific promoters such as the AFP promoter. Specific hypoxia response elements and deleterious genes useful with the method are also as identified above.
  • the invention further includes a substantially isolated polynucleotide having a sequence corresponding to hypoxia response enhancer element(s) (HREE(s)) present in a control region of the muscle pyruvate kinase gene.
  • the element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region.
  • the invention includes an HRE element derived from a muscle pyruvate kinase gene.
  • a substantially isolated polynucleotide having a sequence corresponding to hypoxia response element(s) present in a control region of the endothelin-1 gene.
  • the element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region.
  • the invention includes an HRE element derived from an endothelin-1 gene.
  • Another aspect of the invention includes a substantially isolated polynucleotide having a sequence corresponding to hypoxia response element(s) present in a control region of the enolase 3 (ENO3) gene.
  • the element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region.
  • the invention includes an HRE element derived from an ENO3 gene.
  • FIGS. 1A and 1B show a schematic diagram of the construction of plasmid pGLHRE (FIG. 1B) from plasmid pGL2PV (FIG. 1A).
  • FIGS. 2A, 2B, 2C and 2D show a schematic diagram of the construction of plasmids pGLHSA-150HRE (FIG. 2B), pGL ⁇ MHC 86 -HRE (FIG. 2C), and pGLHCA 118 HRE (FIG. 2D), from plasmid pGLHRE (FIG. 2A).
  • FIGS. 3A and 3B show a schematic diagram of the construction of plasmid pGL ⁇ MHC 1 .2 HRE (FIG. 3B) from plasmid pGLHRE (FIG. 3A).
  • FIGS. 4A and 4B show a schematic diagram of the construction of plasmid pGL ⁇ MHC 1 .2 HRE-NOS (FIG. 4B) from plasmid pGL ⁇ MHC 1 .2 HRE (FIG. 4A).
  • FIGS. 5A and 5B show a schematic diagram of the construction of plasmid p ⁇ MHC 1 .2 -HRE-Bcl-2 (FIG. 5B) from plasmid pSFFV-Bcl-2 (FIG. 5A).
  • FIGS. 6A, 6B, 6C, 6D and 6E show a schematic diagram of the construction of plasmids pGLPKM 460 (FIG. 6C), pGLPKMD (FIG. 6D), and pGLPKM 285 (FIG. 6E) from plasmid pGL2BV (FIG. 6B) and a fragment of the PKM promoter (FIG. 6A; SEQ ID NO:7).
  • FIGS. 7A, 7B and 7C show a schematic diagram of the construction of plasmid pGLET-1 700 (FIG. 7C) from plasmid pGL2BV (FIG. 7B) and a fragment of the ET-1 promoter (FIG. 7A; SEQ ID NO:8).
  • SEQ ID NO:1 is the sense strand nucleotide sequence of a GATA4 enhancer element (Molkentin, et al., 1984).
  • SEQ ID NO:2 is the nucleotide sequence of muscle pyruvate kinase (PKM) sense strand primer F.
  • SEQ ID NO:3 is the nucleotide sequence of PKM reverse strand primer R.
  • SEQ ID NO:4 is the nucleotide sequence of endothelin-1 (Et-1) sense strand primer F.
  • SEQ ID NO:5 is the nucleotide sequence of Et-1 reverse strand primer R.
  • SEQ ID NO:6 is the nucleotide sequence of hypoxia response enhancer element 1 (HREE1), derived from the erythropoietin (EPO) gene (Semenza and Wang), and containing 4 tandem copies of a hypoxia response enhancer (HRE) sequence and cloning linkers.
  • HREE1 hypoxia response enhancer element 1
  • EPO erythropoietin
  • HRE hypoxia response enhancer
  • SEQ ID NO:7 is the nucleotide sequence of a rat muscle pyruvate kinase (PKM) promoter region (Takenaka, et al.).
  • PLM rat muscle pyruvate kinase
  • SEQ ID NO:8 is the nucleotide sequence of a human Et-1 promoter region (Inoue, et al.).
  • SEQ ID NO:9 is the nucleotide sequence of a human cardiac actin promoter region (Minty and Kedes).
  • SEQ ID NO:10 is a nucleotide sequence containing a portion of the rat cardiac ⁇ -myosin heavy chain promoter region (Mahdavi, et al.; GenBank Accession # K01464).
  • SEQ ID NO:11 is a nucleotide sequence containing a portion of the mouse cardiac ⁇ -myosin heavy chain promoter region (Gulick, J., et al.; GenBank Accession # M62404).
  • SEQ ID NO:12 is the nucleotide sequence of a human B-cell leukemia/lymphoma 2 (bcl-2) gene (Tsujimoto, et al.; GenBank Accession # M13994).
  • SEQ ID NO:13 is the predicted amino acid sequence from SEQ ID NO:12.
  • SEQ ID NO:14 is the nucleotide sequence of a rat nitric oxide synthase (bNOS) gene (Bredtet al.; EMBL Accession # X59949).
  • SEQ ID NO:15 is the predicted amino acid sequence from SEQ ID NO:14.
  • SEQ ID NO:16 is the nucleotide sequence of a human bcl-2 fusion gene (Seto, et al.; EMBL Accession # X06487).
  • SEQ ID NO:17 is the predicted amino acid sequence from SEQ ID NO:16.
  • SEQ ID NO:18 is the nucleotide sequence of a human NOS-1 gene (Fujisawa, et al.); DDBJ Accession # D16408; NCBI Seq ID 506339)
  • SEQ ID NO:19 is the predicted amino acid sequence from SEQ ID NO:18.
  • SEQ ID NO:20 is the nucleotide sequence of a human NOS-SN gene (Nakane, et al.; GenBank Accession # L02881)
  • SEQ ID NO:21 is the predicted amino acid sequence from SEQ ID NO:20.
  • SEQ ID NO:22 is the nucleotide sequence of a 256 base pair (bp) 3' EPO-1 hypoxia response enhancer element (Semenza and Wang).
  • SEQ ID NO:23 is the nucleotide sequence of a 42 bp 3' EPO-1 hypoxia response enhancer element (Madan, et al.).
  • SEQ ID NO:24 is the nucleotide sequence of an 86 bp rat ⁇ MHC promoter region.
  • SEQ ID NO:25 is the nucleotide sequence of a mouse catalase gene (Reimer, et al.; GenBank # L25069).
  • SEQ ID NO:26 is the predicted amino acid sequence from SEQ ID NO:25.
  • SEQ ID NO:27 is the nucleotide sequence of a human manganese superoxide dismutase (SOD) gene (Clair, et al.; EMBL # X59445).
  • SEQ ID NO:28 is the predicted amino acid sequence from SEQ ID NO:27.
  • SEQ ID NO:29 is the nucleotide sequence of a human ⁇ -enolase (ENO3) gene (Giallongo, et al.; EMBL # X56832) between nucleotides -628 to +63.
  • ENO3 human ⁇ -enolase
  • SEQ ID NO:30 is the predicted amino acid sequence from SEQ ID NO:29.
  • SEQ ID NO:31 is a consensus sequence of a region present in both the PKM and ENO3 promoters.
  • Ischemia is defined as an insufficient supply of blood to a specific organ or tissue. A consequence of decreased blood supply is an inadequate supply of oxygen to the organ or tissue (hypoxia). Prolonged hypoxia may result in injury to the affected organ or tissue. "Anoxia” refers to a virtually complete absence of oxygen in the organ or tissue, which, if prolonged, may result in death of the organ or tissue.
  • Hypoxic condition is defined as a condition under which a particular organ or tissue receives an inadequate supply of oxygen.
  • Anoxic condition refers to a condition under which the supply of oxygen to a particular organ or tissue is cut off.
  • Reperfusion refers to the resumption of blood flow in a tissue following a period of ischemia.
  • Ischemic injury refers to cellular and/or molecular damage to an organ or tissue as a result of a period of ischemia and/or ischemia followed by reperfusion.
  • hypoxia response enhancer element when used in the context of nucleic acid constructs, refers to a region of the construct or a nucleic acid fragment having a defined function.
  • a hypoxia response enhancer element is a region of DNA that, when associated with a gene operably linked to a promoter, enhances the transcription of that gene under hypoxic conditions.
  • operably linked denotes a relationship between a regulatory region (typically a promoter element, but may include an enhancer element) and the coding region of a gene, whereby the transcription of the coding region is under the control of the regulatory region.
  • Two nucleic acid elements are said to be "heterologous” if the elements are derived from two different genes, or alternatively, two different species.
  • a hypoxia response enhancer element from a human erythropoietin gene is heterologous to a promoter from a human myosin gene.
  • a hypoxia response enhancer element from a human erythropoietin gene is heterologous to a promoter from a mouse erythropoietin gene.
  • Control region refers to specific sequences at the 5' and 3' ends of eukaryotic genes which may be involved in the control of either transcription or translation.
  • most eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription initiation site.
  • most eukaryotic genes have a CXCAAT region (X may be any nucleotide) 70 to 80 bases upstream from the start of transcription.
  • AATAAA sequence which may be the signal for addition of the polyadenylation tail to the 3' end of the transcribed mRNA.
  • Chimeric gene also termed “chimeric DNA construct” refers to a polynucleotide containing heterologous DNA sequences, such as promoter and enhancer elements operably linked to a therapeutic gene.
  • a construct containing a human ⁇ -myosin heavy chain ( ⁇ -MHC) promoter fragment operably linked to a human bcl-2 gene and containing a human erythropoietin gene hypoxia response element comprises an exemplary chimeric gene.
  • the present invention relates to chimeric genes having at least three functional elements: (i) a therapeutic gene, (ii) a tissue-specific promoter, and (iii) a hypoxia response enhancer (HRE) element.
  • the tissue-specific promoter in combination with the HRE element directs expression of the therapeutic gene in a selected tissue under hypoxic conditions.
  • the gene is preferably introduced into a target tissue as part of a complete expression vector in a pharmaceutically-acceptable vehicle, either by direct administration to the target tissue (e.g., injection into the target tissue), or by systemic administration (e.g., intravenous injection).
  • the gene may be targeted to a selected tissue, for example, by incorporating it in a virion expressing a modified envelope protein designed to bind to receptors preferentially expressed on cells from the selected, or targeted, tissue.
  • expression of the gene in tissues other than the target tissue, and under conditions other than hypoxic or anoxic is preferably minimal.
  • a promoter in the context of the present specification, refers to a polynucleotide element capable of regulating the transcription of a gene adjacent and downstream (3') of the promoter.
  • the promoter may contain all of, or only a portion of, the complete 5' regulatory sequences of the gene from which it is derived.
  • a sequence in the promoter region is typically recognized by RNA polymerase molecules that start RNA synthesis.
  • a promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular tissue. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-specific factor), under certain conditions (e.g., hypoxia, or the presence of an enhancer element in the chimeric gene containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult).
  • tissue-specific factor e.g., a tissue-specific factor
  • Promoters useful in the practice of the present invention are preferably tissue-specific--that is, they are capable of driving transcription of a gene in one tissue while remaining largely “silent” in other tissue types. It will be understood, however, that tissue-specific promoters may have a detectable amount of "background” or “base” activity in those tissues where they are silent.
  • the degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue).
  • a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 5. Preferably, the selectivity ratio is greater than about 15.
  • promoters While not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific", and are contemplated for use with the present invention. For example, promoters that are active in a variety of central nervous system (CNS) neurons may be therapeutically useful in protecting against damage due to stroke, which may effect any of a number of different regions of the brain.
  • CNS central nervous system
  • Tissue-specific promoters may be derived, for example, from promoter regions of genes that are differentially expressed in different tissues. For example, a variety of promoters have been identified which are suitable for upregulating expression in cardiac tissue. Included are the cardiac ⁇ -myosin heavy chain (AMHC) promoter and the cardiac ⁇ -actin promoter.
  • AHC cardiac ⁇ -myosin heavy chain
  • promoters useful in the present invention possess a relatively low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissues.
  • One means of achieving this is to select promoters of genes encoding proteins that have a relatively low turnover rate in adult tissue, such as the actin and ⁇ -MHC promoters described herein.
  • Another means is to use "silencer" elements, which suppress the activity of a selected promoter in the absence of hypoxia.
  • the level of expression of a gene under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis). Vectors used for such experiments typically contains a reporter gene, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities.
  • This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring hypoxia sensitivity.
  • tissue specific promoters may be particularly advantageous in practicing the present invention. In most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector.
  • promoter fragments may be isolated using the polymerase chain reaction (PCR; Mullis, Mullis, et al.). Cloning of amplified fragments may be facilitated by incorporating restriction sites at the 5' ends of the primers.
  • PCR polymerase chain reaction
  • Promoters suitable for cardiac-specific expression include the promoter from the murine cardiac ⁇ -myosin heavy chain gene.
  • the gene contains a 5.5 kbp promoter region which may be obtained as a 5.5 kbp SacI/Sall fragment from the murine ⁇ MHC gene (Subramaniam, et al., 1991).
  • Reporter gene constructs utilizing this 5.5 kbp ⁇ MHC promoter are expressed at relatively high levels selectively in cardiac tissue (whether or not an HREE is present) and, when present in transgenic animals, are regulated in a similar fashion to the endogenous gene (Subramaniam, et al., 1991).
  • a smaller fragment of the rat ⁇ -MHC promoter may be obtained as a 1.2 kbp EcoRI/HindIII fragment (Gustafson, et al.).
  • constructs utilizing the 1.2 kbp rat ⁇ MHC promoter are expressed at a low level in the absence of an HREE, and at an intermediate level in the presence of an HREE.
  • the ⁇ MHC 1 .2 promoter is an exemplary promoter to target expression of chimeric genes of the present invention to cardiac tissue. Expression of genes under the control of this promoter fragment is very low in cardiac cells under normal oxygenation conditions, but is increased by about a factor of four under hypoxic conditions when the construct contains HREE1. Expression in cells other than cardiac cells is at background levels.
  • An 86 bp fragment of the rat ⁇ MHC promoter restricts expression of reporter genes to cardiac and skeletal muscle (i.e., it has lost some tissue selectivity). Additional cardiac specificity may be conferred to the fragment by ligating (e.g., blunt end ligating) a 36-mer oligonucleotide (SEQ ID NO:1) containing cardiac-specific GATA4 enhancer elements just upstream of base pair -86 (Molkentin, et al., 1984). This promoter fragment also results in low levels of expression in the absence of additional enhancers such as HRE elements. The low level of basal expression induced by the 86 bp fragment, and the ability to upregulate this basal level of expression with a hypoxia response enhancer element are useful properties for a promoter for use with the present invention.
  • cardiac-specific promoter regions from the rat and mouse AMHC genes are presented herein as SEQ ID NO:10 and SEQ ID NO:11, respectively. Both sequences end just upstream of the ATG initiation codons of their respective genes.
  • Other cardiac-specific promoters include the cardiac ⁇ -actin promoter and the myosin light chain-2 (MLC-2) promoter. Constructs described herein utilizing a 118 bp fragment (SEQ ID NO:9) from the human cardiac ⁇ -actin (HCA) promoter result in a relatively low level of cardiac-specific expression, which may be increased by the inclusion of an HREE in the expression construct (Example 1, Table 1).
  • the cardiac-specific myosin light chain-2 promoter may be obtained as a 2.1 kbp KpnI/EcoRI fragment from the rat cardiac myosin light chain-2 (MLC-2) gene (Franz, et al.).
  • Prostate-specific promoters include the 5'-flanking regions of the human glandular kallikrein-1 (hKLK2) gene and the prostate-specific antigen (hKLK3; PSA) gene (Murtha, et al.; Luke, et al.).
  • the renin promoter is suitable for directing kidney-specific expression (Fukamizu, et al.), while the aldolase-C promoter (Vibert, et al.) or the tyrosine hydroxylase promoter (Sasaoka, et al.) may be used to direct expression in the brain.
  • Promoters specific for vascular endothelium cells include the Et-1 promoter (Inoue, et al.) and vonWillebrand factor (Robondi and Lynch) promoter.
  • Tumor-specific promoters include the ⁇ -fetoprotein (AFP) promoter, contained in a 7.6 kbp fragment of 5'-flanking DNA from the mouse AFP gene (Marci, et al.). This promoter normally directs expression of the AFP gene in fetal liver and is transcriptionally silent in adult tissues. However, it can be abnormally reactivated in hepatocellular carcinoma (HCC), conferring tumor-specific expression in adult tissue (Marci, et al.).
  • HCC hepatocellular carcinoma
  • promoters are exemplary promoters for use with the present invention.
  • Other promoters suitable for use with the present invention may be selected by one of ordinary skill in the art following the guidance of the present specification.
  • Therapeutic genes contained in constructs of the present invention are preferably expressed at low levels, if at all, under conditions of normal oxygenation (minimizing any side effects). Under conditions of hypoxia, however, expression of the genes is increased, affording protection to the target tissue. The elevated expression under hypoxic conditions is conferred by the presence of one or more hypoxia response enhancer (HRE) elements.
  • HRE hypoxia response enhancer
  • HRE elements contain polynucleotide sequences that may be located either upstream (5') or downstream (3') of the promoter and/or therapeutic gene.
  • the HRE element (HREE) is typically a cis-acting element, usually about 10-300 bp in length, that acts on a promoter to increase the transcription of a gene under the control of the promoter.
  • the promoter and enhancer elements are selected such that expression of a gene regulated by those elements is minimal in the presence of a healthy supply of oxygen, and is upregulated under hypoxic or anoxic conditions.
  • Hypoxia response enhancer elements are found in association with a number of genes, including the erythropoietin (EPO) gene.
  • EPO erythropoietin
  • Exemplary HRE elements from the EPO gene are presented herein as SEQ ID NO:6, SEQ ID NO:22 and SEQ ID NO:23.
  • SEQ ID NO:22 results in approximately a five-fold induction of reporter gene expression under hypoxic conditions (Semenza and Wang), while, the element having the sequence represented as SEQ ID NO:23 results in approximately a 17-fold increase in activity under hypoxic conditions (Madan, et al.)
  • Example 1 Experiments performed in support of the present invention (e.g., Example 1) demonstrate that expression of constructs containing HREE1 (SEQ ID NO:6) is increased by approximately 5- to 7-fold in response to hypoxic conditions. These results indicate that the HREE1 element is fully functional when fused to muscle and cardiac specific promoters and that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters.
  • constructs containing a fragment (SEQ ID NO:29) from the control region of the enolase 3 (ENO3) gene was induced approximately 5 to 8 fold by hypoxia in C2C12 cells and cardiac myocytes respectively (see Table 1).
  • HREE in the ENO3 promoter fragment may be a particularly effective HREE for hypoxia induction in constructs containing a tissue-specific promoter, such as a cardiac or skeletal muscle promoter.
  • exemplary hypoxia response enhancer elements may also be isolated from regulatory regions of both the muscle glycolytic enzyme pyruvate kinase (PKM) gene (Takenaka, et al.), the human muscle-specific ⁇ -enolase gene (ENO3; Peshavaria and Day), and the endothelin-1 (ET-1) gene (Inoue, et al.).
  • PKM muscle glycolytic enzyme pyruvate kinase
  • ENO3 human muscle-specific ⁇ -enolase gene
  • ET-1 endothelin-1
  • a portion of this HRE element obtained by digesting with SmaI to cut at an internal SmaI site, localized the hypoxia response sequence to a 200 bp fragment.
  • This fragment termed HREPKM 285 , confers hypoxia-induced expression in C2C12 myotubes and cardiac myocytes that is at least equivalent to that obtained using HREE1 (SEQ ID NO:6).
  • Both PKM and ENO3 promoters contain a common sequence element (SEQ ID NO:31) located at 5' -88 and -70 bp respectively from the transcription start sites.
  • SEQ ID NO:31 a common sequence element located at 5' -88 and -70 bp respectively from the transcription start sites.
  • An oligonucleotide containing this sequence may be sufficient to confer hypoxia response characteristics to constructs of the present invention.
  • pGLET-1 700 containing 700 bp of the human ET-1 gene promoter (SEQ ID NO:8), in transfected human arterial endothelial cells was increased approximately 5 -fold by incubation of the cells in a hypoxic atmosphere. No hypoxia-induced increase in pGLET-1 700 expression was seen in other cell types, including HeLa cells, C2C12 cells, and cardiac myocytes.
  • the 700 bp fragment may be used to target hypoxia regulated genes specifically to cells of the vascular endothelium, since the fragment contains element(s) conferring tissue specificity (i.e., elements effective to target expression exclusively to the vascular endothelium), as well as HRE element(s) effective to upregulate transcription of a gene under control of the fragment during hypoxic conditions.
  • element(s) conferring tissue specificity i.e., elements effective to target expression exclusively to the vascular endothelium
  • HRE element(s) effective to upregulate transcription of a gene under control of the fragment during hypoxic conditions.
  • HRE elements not explicitly identified above. Additional HRE elements may be identified, for example, as detailed in Examples 4 and 5. Further, promoter deletion and mutation analyses (e.g., as described above and in Webster and Kedes) may be used to identify such elements in other hypoxia responsive genes. A number of such responsive target genes have been shown to be induced when cells are exposed to hypoxia in vitro (e.g., Heakock and Sutherland).
  • tissue-specific promoter and the hypoxia response enhancer element(s) of the present invention may be derived from a contiguous polynucleotide sequence from a single gene (e.g., as shown above for the ET-1 promoter region, which contains HRE element(s) and also imparts endothelial cell-specific expression).
  • the present invention may be used to alleviate a number of disease conditions resulting from hypoxic and/or anoxic conditions due to ischemia where cell and tissue damage results from ischemia and ischemia followed by reperfusion.
  • the invention is particularly suitable in cases where the subject is diagnosed to be at risk for an ischemic episode in a particular tissue.
  • constructs capable of expressing therapeutic genes into their cardiac tissue in order to decrease the risk of injury to the tissue during any subsequent ischemic episodes.
  • Such constructs may serve to protect, for example, cardiac and vascular endothelial tissues from ischemic damage and thereby prevent the progression of the heart disease.
  • Recurrent ischemia and reperfusion typically results in oxidative damage to cells from reactive oxygen species (free radicals), such as peroxides, that are generated during redox switching (Frei).
  • reactive oxygen species free radicals
  • peroxides reactive oxygen species
  • contact of fresh blood with damaged or dead cells induces the influx of neutrophils, or pus cells, which kill heart cells which would otherwise have recovered.
  • Much of the damage caused by neutrophils has been attributed to superoxide ions.
  • the superoxide anion can damage tissue in several ways.
  • the interaction of the superoxide anion with hydrogen peroxide leads to the production of hydroxyl radicals which are potentially toxic and react rapidly with most organic molecules. Lipids, proteins, and nucleic acids may all be primary targets for such oxidative damage.
  • the extent and type of damage depend on the severity and nature of the hypoxic stress. For example, the stress may cause cellular damage, initiating an inflammatory response with neutrophil attack and subsequent tissue necrosis. Alternatively, the stress may initiate
  • tissue death occurs (necrosis or apoptosis)
  • the damage caused by ischemia-reperfusion episodes is typically the result of redox reactions and is quantitatively related to the severity and duration of the ischemia.
  • a severe heart attack may result in extensive damage (e.g., infarction of 30% to 40% of the left ventricle), whereas moderate angina and silent repetitive ischemia may result in relatively minor damage during each episode.
  • anti-oxidant systems that may intervene immediately at the sites of intracellular redox reactions to minimize damage
  • vasodilator systems that may minimize the severity of the ischemia by increasing blood flow to vulnerable tissues.
  • Antioxidant proteins amenable for use with the present invention include gene products of Bcl-2, catalase and superoxide dismutase (SOD) genes, while proteins with vasodilative properties include nitric oxide synthase (NOS), which produces the vasodilator nitric oxide (NO).
  • NOS nitric oxide synthase
  • Bcl-2 an integral inner mitochondrial membrane protein of relative molecular mass ⁇ 25 kDa, has been shown to protect certain cells against apoptosis (Hockenbery, et al., 1990) by acting as an antioxidant (Hockenbery, et al., 1993).
  • Bcl-2 may be an effective therapeutic gene for reducing damage to tissues during ischemic episodes because apoptosis may be a common response of many tissues, including the heart, to oxidative stress (Williams and Smith; Gottling, et al.
  • the enzyme superoxide dismutase catalyzes the decomposition of the superoxide anion to peroxide.
  • Enzymes such as superoxide dismutase, free radical scavengers or agents which prevent the influx on neutrophils are able to increase the salvage of heart muscle cells.
  • the enzyme catalase in turn catalyzes the conversion of peroxides to water.
  • Exemplary sequences of a SOD gene and a catalase gene are presented herein as SEQ ID NO:27 and SEQ ID NO:25, respectively.
  • the sequence presented herein as SEQ ID NO:27 encodes a manganese SOD, which has a relatively long half-life.
  • a related sequence, of a human Cu/Zn SOD may be found in Gorechi, et al. The Cu/Zn SOD has a shorter half-life than the manganese SOD.
  • Endothelial-derived nitric oxide regulates the expression of vasoconstrictors and growth factors by the vascular endothelium (Kourembanas, et al.).
  • endothelial cells typically increase expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor. This increase in expression can be reduced or prevented by exposure to NO (Kourembanas, et al.).
  • ET-1 endothelin-1
  • One of the effects of ET-1 induced vasoconstriction is decreased blood flow to the affected organ or tissue, which can exasperate hypoxic damage due to ischemia. According to the present invention, such damage may be reduced by providing NO to the affected tissue through the expression of a NOS gene under the control of a vascular epithelium or cardiac-specific promoter and hypoxia response enhancer element.
  • Therapeutic genes of the present invention may be preferably derived from the same or related species as the one to which the methods and compositions of the present invention are applied. For example, for therapeutic treatment of a dog, it may be desirable to utilize a construct containing a therapeutic gene cloned from a dog. Similarly, for treatment of human conditions, it may be desirable to utilize therapeutic genes cloned from human-derived nucleic acids.
  • genes encoding the proteins discussed above represent exemplary therapeutic genes useful in the practice of the present invention. It will be appreciated, however, that following the teachings and guidance of the present specification, one of skill in the art may select other therapeutic genes effective to reduce cellular damage due to hypoxia or ischemia, and that the use of such genes is considered to be within the scope of the present invention.
  • the present invention includes constructs containing deleterious genes, rather than therapeutic genes.
  • Expression of the deleterious genes is targeted to tissues which are harmful (e.g., malignant tumors) or otherwise undesirable. Promoters and hypoxia response elements may be selected as described above. Promoters useful in this aspect of the invention preferably restrict expression only to the undesirable tissue.
  • the AFP promoter can be activated in hepatocellular carcinoma (HCC), conferring tumor-specific expression in adult tissues (Marci, et al.).
  • Deleterious genes include a viral thymidine kinase gene (tk), such as the herpes simplex virus (HSV) tk. This gene is not deleterious by itself, but when expressed, viral TK can phosphorylate ganciclovir (GCV), turning GCV into a cytotoxic compound. Since tumor cells are typically hypoxic, constructs having a tumor-specific promoter operably linked to a viral tk and an HREE may be used in conjunction with GCV to selectively kill tumor cells.
  • tk viral thymidine kinase gene
  • HSV herpes simplex virus
  • Chimeric genes of the present invention are preferably incorporated into expression vectors capable of expressing a therapeutic gene product in a selected eukaryotic host cell (i.e., a target tissue).
  • expression vectors may contain, in addition to the chimeric gene, various other sequences useful for effective expression of the therapeutic gene in selected tissues.
  • sequences may include, for example, sequences necessary for the termination of transcription. These sequences are transcribed as polyadenylated segments in the untranslated portion of the MRNA encoding the desired therapeutic protein. The 3' untranslated regions may also include transcription termination sites.
  • Vector constructs made in support of the present invention are designed to express either a reporter gene (e.g., luciferase), or therapeutic genes (e.g., Bcl-2 or NOS).
  • Therapeutic gene expression is under the control of either a ubiquitous promoter (e.g., SV40), or a tissue-specific promoter (e.g., striated muscle or cardiac-specific promoter).
  • hypoxia response enhancer elements
  • EPO erythropoietin
  • PLM muscle specific pyruvate kinase
  • ENO3 enolase 3
  • Et-1 endothelial cell endothelin-1
  • the data shown in Table 1 demonstrate that cells containing constructs having a hypoxia response enhancer element, such as HREE1, in combination with a compatible promoter, express the reporter at levels that are 5 to 7 times greater under hypoxic conditions than under aerobic conditions, and that HREE1 is equally active in different cells and independent of the promoter.
  • the data also demonstrate that expression of constructs containing ⁇ -MHC promoters is cardiac specific, and that the basal (aerobic) expression from ⁇ -MHC 1 .2 and HCA promoters is relatively low. Further, the data indicate that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters.
  • hypoxia-induced expression may peak significantly earlier than 20 hours, and (ii) repeat ischemic episodes may upregulate expression more the single experimental episode used herein. Accordingly, the 2-fold induction may be an underestimate of the level of enhancement of transcription/expression caused by ischemia.
  • Example 3 In vitro experiments (Example 3) demonstrate that cells transfected with reporter (pGLHRE, pGLHCA 118 HRE, pGL ⁇ MHC 1 .2 HRE) and therapeutic (pSFFV-Bcl-2 and pNOS-HRE) constructs appear normal and respond to stimuli as expected. Reporter-transfected cells differentiate normally and respond to hypoxia with the predicted induction of reporter, while NOS and bcl-2-transfected cells appear normal both during the hypoxia and during subsequent reoxygenation. These results suggest that inclusion of HRE elements, Bcl-2 over-expression, and hypoxia-induced over-expression of NOS is not toxic or deleterious to muscle cells in vitro.
  • expression vectors carrying therapeutic genes of the present invention may be effective to protect tissues from ischemic damage.
  • Such protective effects may be assayed in an animal model by, for example, infecting myocardial tissue with an expression vector containing a chimeric gene of the present invention, such as an adenoviral vector expressing a therapeutic gene (e.g., Bcl-2 or SOD), a cardiac-specific promoter, and an HRE element, as described, for instance, in Example 2.
  • a chimeric gene of the present invention such as an adenoviral vector expressing a therapeutic gene (e.g., Bcl-2 or SOD), a cardiac-specific promoter, and an HRE element, as described, for instance, in Example 2.
  • the animals may be subjected to repeat ischemic episodes (e.g., 30 minutes to 1 hour) followed by reperfusion (e.g., 1 to 8 hours). Following the last reperfusion, the animals may be sacrificed and the ischemic regions of the myocardium may be tested for the presence and extent of infarction as described, for example, by Thornton, et al., and for the presence of apoptosis as described, for example, in Gottlieb, et al. Sample biopsies may also be assayed for expression of the therapeutic gene by Northern blots.
  • any of a variety of methods known to those skilled in the art may be used to introduce chimeric genes of the present invention into selected target tissue cells.
  • gene therapy of cardiac tissue has included lipofection, retrovirus and adenovirus-mediated gene transfer, and injection of naked DNA directly into the vascular endothelium or cardiac tissue (Nabel, et al.; Lin, et al.; Leclere, et al.; Flugelman, et al.). These and other methods are discussed more fully in the sections below.
  • Host cells may be transfected with chimeric genes of the present invention by infection with mature virions containing hybrid vectors (the chimeric genes along with selected viral sequences).
  • the virions used to transfect host cells are preferably replication-defective, such that the virus is not able to replicate in the host cells.
  • the virions may be produced by co-infection of cultured host cells with a helper virus. Following coinfection, the virions are isolated (e.g., by cesium chloride centrifugation) and any remaining helper virus is inactivated (e.g., by heating).
  • the resulting mature virions contain a chimeric gene of the present invention and may be used to infect host cells in the absence of helper virus.
  • high titers of replication-defective recombinant virus, free of helper virus may be produced in packaging cell lines containing those components for which the virus is defective (Miller).
  • viruses including retroviruses, adeno-associated virus (AAV), herpes virus, vaccinia virus, and several RNA viruses may be amenable for use as vectors with chimeric gene constructs of the present invention.
  • AAV adeno-associated virus
  • vaccinia virus a virus that has specific advantages and disadvantages, which are appreciated by those of skill in the art.
  • Methods for manipulating viral vectors are also known in the art (e.g., Grunhaus and Horowitz; Hertz and Gerard; and Rosenfeld, et al.)
  • Retroviruses like adeno-associated viruses, stably integrate their DNA into the chromosomal DNA of the target cell. Unlike adeno-associated viruses, however, retroviruses typically require replication of the target cells in order for proviral integration to occur. Accordingly, successful gene transfer with retroviral vectors depends on the ability to at least transiently induce proliferation of the target cells.
  • Retroviral vectors are attractive in part due to the efficiency of transfection--some vectors can stably transduce close to 100% of target cells.
  • the use of retroviral vectors for in vivo gene therapy has been limited, in part, by the requirement of appropriate viral receptors on the target cell. Because the identities of most retroviral receptors are unknown, it has not been possible to determine the distribution of receptors in different cell types. Accordingly, the targeting of specific cell types by retroviral vectors has in many cases proven problematic.
  • This difficulty may be circumvented by modifying the envelope protein of the retrovirus to contain a ligand for a known endogenous (not necessarily viral) receptor expressed on the target cells.
  • a ligand for a known endogenous (not necessarily viral) receptor expressed on the target cells An application of this technique is described in detail by Kasahara.
  • the virus also contains an unmodified envelope protein to facilitate cell entry.
  • a number of receptors, such as desmin, E-selectin, and A-CAM are expressed preferentially on cardiac cells and may be amenable to this approach (e.g., Hansen and Stawaski; Lefer, et al.; Youker, et al.).
  • Adeno-associated viruses are capable of efficiently infecting nondividing cells and expressing large amounts of gene product. Furthermore, the virus particle is relatively stable and amenable to purification and concentration. Replication-defective adenoviruses lacking portions of the E1 region of the viral genome may be propagated by growth in cells engineered to express the E1 genes (Jones and Shenk; Berkner; Graham and Prevea). Most of the currently-used adenovirus vectors carry deletions in the E1A-E1B and E3 regions of the viral genome.
  • Herpes virus vectors (Breakefield and DeLuca; Freese, et al.) are particularly well suited for the delivery and expression of foreign DNA in cells of the central nervous system (CNS), since they can efficiently infect mature, postmitotic neurons.
  • Methods for manipulating the vectors and transfecting CNS cells are well known (see, e.g., Kennedy and Steiner; Yung).
  • a number of studies describe methods for transplanting genetically modified cells into different regions of the brain (Malim, et al.; Rossi and Sarver; Sullenger, et al.; Morgan, et al.; Chatterjee, et al.; Malin, et al.; Hope, et al.).
  • Studies utilizing direct injection of vectors into CNS tissue have also been performed (e.g., Zhang, et al.).
  • Plasmids bearing chimeric genes of the present invention may be purified and injected directly into a target tissue, as exemplified in Example 2 for rat cardiac tissue.
  • the data discussed in Example 2 demonstrate that cardiac injection of plasmid suspended in saline buffer is effective to result in expression of the plasmid in the cardiac cells.
  • Similar approaches have been used successfully by others to express, for example, exogenous genes in rodent cardiac and skeletal muscle (Wolf, et al.; Ascadi, et al., 1991a; Ascadi, et al., 1991b; Lin, et al.; Kitsis, et al..
  • Liposomes may be employed to deliver genes to target tissues using methods known in the art.
  • the liposomes may be constructed to contain a targeting moiety or ligand, such as an antigen, an antibody, or a virus on their surface to facilitate delivery to the appropriate tissue.
  • a targeting moiety or ligand such as an antigen, an antibody, or a virus on their surface to facilitate delivery to the appropriate tissue.
  • liposomes prepared with ultraviolet (UV) inactivated Hemagglutinating Virus of Japan (HVJ) may be used to deliver DNA to selected tissues (Morishita, et al.).
  • UV ultraviolet
  • HVJ Hemagglutinating Virus of Japan
  • the liposomes may also be surface-coated, e.g., by incorporation of phospholipid--polyethyleneglycol conjugates, to extend blood circulation time and allow for greater targeting via the bloodstream. Liposomes of this type are well known.
  • Receptor-mediated endocytic pathways for the uptake of DNA may permit the targeted delivery of genes to specific cell types in vivo.
  • Receptor-mediated methods of gene transfer involve the generation of complexes between plasmid DNA and specific polypeptide ligands (Wu) that can be recognized by receptors on the cell surface.
  • Wu polypeptide ligands
  • One of the problems with receptor-mediated uptake for gene delivery is that the endocytic vesicles formed during this process may be transported to the lysosome, where the contents of the endosome are degraded. Methods have been developed to facilitate escape of the DNA from the endosome during the course of its transport.
  • either whole adenovirus (Wagner, et al., 1992a; Christiano, et al.) or fusogenic peptides of the influenza HA gene product (Wagner, et al., 1992b) may be used to induce efficient disruption of DNA-containing endosomes.
  • a vector may be targeted to selectively transfect a specific population of cells
  • the vector in addition to local administration (such as may be achieved by injection into the target tissue), the vector may be administered systemically (e.g., intravenously) in a biologically-compatible solution or pharmaceutically acceptable delivery vehicle.
  • Vector constructs administered in this way may selectively infect the target tissue.
  • the presence of a target tissue-specific promoter on the construct provides an independent means of restricting expression of the therapeutic gene.
  • compositions and methods of the present invention may be useful to prevent tissue damage and/or death, due to ischemia and/or subsequent reperfusion, in a variety of tissues.
  • an exemplary application is in the reduction of damage due to recurrent myocardial ischemia following a heart attack.
  • the expression of therapeutic genes in the cardiac tissue of heart attack victims may decrease the risk of injury to the tissue during any subsequent ischemic episodes.
  • subjects who have been diagnosed with transient cerebral ischemia, blood clots or other risk factors for stroke may benefit from the use of hypoxia-inducible brain-specific constructs.
  • Subjects diagnosed with acute or chronic renal failure are at greater risk for further ischemic damage to the kidneys (e.g., Rosenberg and Paller).
  • Such subjects may benefit from a therapeutic gene under the control of a kidney-specific promoter, expression of which is enhanced by hypoxic conditions.
  • a variety of other tissues diagnosed as "at risk" for ischemia may be similarly protected, as will be appreciated by one of skill in the art having the benefit of the present specification.
  • compositions e.g., expression vectors containing chimeric genes of the present invention
  • methods of the present invention also have a number of applications in animal medicine. Although animals do not usually develop classical atherosclerosis, cardiomyopathies are very common. A number of species develop ischemia-related syndromes, including arteritis, vasculitis, and related vasculopathies, that result in direct redox damage to cells and tissues, particularly to vascular walls and myocardial tissues. Such conditions may be alleviated by administration of chimeric genes of the present invention.
  • a common and serious condition in horses and ponies involves ascending colonic ischemia, usually caused by strangulation obstruction (Dabareiner, et al.; Sullivan, et al.; Wilson and Stick).
  • strangulation obstruction Danger, et al.
  • Sullivan, et al. Wilson and Stick
  • a related disease in dogs is called gastric dilation-volvulus (Lantz, et al.).
  • Treatment of these disorders typically involves surgical removal of the obstruction.
  • Reperfusion following such surgery can result in significant injury to reperfused tissues, and typically triggers an inflammatory response with progressive tissue necrosis. The reperfusion may also results in death of the animal due to cardiogenic shock.
  • Compositions and methods of the present invention may be used therapeutically to treat such conditions, and to provide protection to vulnerable tissues, including heart and vascular endothelium, during the treatment of the above syndromes.
  • Another utility of the present invention is the treatment of cardiac disease in cats and dogs (Miller, et al.).
  • a variety of forms of cardiovascular disease have been described in both cats and dogs, including dilated cardiomyopathy, left ventricular hypertrophy, and hyperthyroidism (Fox, et al.; Atkins, et al.).
  • Systemic necrotizing vasculitis a condition that may be analogous to atherosclerosis in humans (with regard to plaque formation and intimal proliferation), has been described in Beagles (Scott-Moncrieff, et al.). Each of these conditions may involve ischemia and reperfusion redox injuries to cardiac and vascular tissue that may be treated using the methods and compositions of the present invention.
  • the present invention may also be employed in diagnostic applications, where it is desirable to localize the site of hypoxia or anoxia.
  • therapeutic genes are replaced by reporter genes, such as those used in experiments performed in support of the present invention (e.g., luciferase).
  • reporter genes such as those used in experiments performed in support of the present invention (e.g., luciferase).
  • the chimeric genes containing the reporter genes under the control of a selected promoter and a hypoxia response element are introduced into a tissue where it is desirable to localize the site of hypoxia. Hypoxia is localized by increased expression of the reporter gene.
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.
  • transformation methods are commonly used in the art, and may be found, for example, in Ausubel, et al., and Sambrook, et al.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO 4 and electroporation. Successful transfection is generally recognized when any indication of the operation of the expression vector occurs within the host cell.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences (restriction sites) in the DNA.
  • the various restriction enzymes used herein are commercially available (e.g., New England Biolabs, Beverly, Mass.) and their reaction conditions are known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of a plasmid or of a DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • Ligation refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (e.g., Sambrook, et al.). Unless otherwise noted, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • “Filling” or “blunting” refer to the procedures by which the single stranded end in the cohesive terminus of a restriction enzyme-cleaved nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a versatile tool for converting a restriction cut end that may be cohesive with the ends created by only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuclease or other filled cohesive terminus.
  • blunting is accomplished by incubating 2-15 ⁇ g of the target DNA in a buffer containing 10 mM MgCl 2 , 1 Mm dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) at about 37° C. in the presence of 8 units of the Klenow fragment of DNA polymerase I (Boehringer Mannheim, Indianapolis, Ind.) and 250 ⁇ M of each of the four deoxynucleoside triphosphates (Boehringer Mannheim). The incubation is generally terminated after about 30 min.
  • the reaction products may be purified using standard phenol and chloroform extraction methods followed by ethanol precipitation.
  • Northern blotting is a method by which the presence of a cellular MRNA is confirmed by hybridization to a known, labelled oligonucleotide, DNA or RNA fragment.
  • Northern analysis shall mean electrophoretic separation of RNA, typically MRNA, on agarose (e.g., 1%) in the presence of a denaturant (e.g., 7% formaldehyde), transfer to nitrocellulose or nylon membrane, hybridization to the labelled fragment, washing, and detection of the labeled fragment, as described by Sambrook, et al.
  • HeLa cells, Hep G2 cells and C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC; Rockville, Md.). Human arterial endothelial cells were obtained from Clonetics Corp. (San Diego, Calif.). Unless otherwise indicated, the cells were grown at 37° C. under 5 or 10% CO 2 in MEM or DMEM medium (Gibco/BRL) containing 10% fetal bovine serum (Gibco/BRL).
  • Cardiac myocytes were isolated and cultured as described previously (Bishopric, et al., Webster and Bisphopric, 1992). Briefly, hearts from about 30 (three litters) were minced and subjected to serial trypsin digestion to release single cells. After the final digestion, the cells were washed and preplated for 0.5 h in minimal essential medium (MEM; Gibco/BRL, Gaithersburg, Md.) with 5% fetal calf serum (FCS; Gibco/BRL).
  • MEM minimal essential medium
  • FCS fetal calf serum
  • Nonattached cells were re-plated in 60-mm Falcon dishes (Becton Dickinson Labware, Lincoln Park, N.J.) at a density of about 2.5 ⁇ 10 6 cells per dish in MEM containing 5% fetal calf serum, 2.0 g/l glucose and 10 mM HEPES, and grown at 37° C. under 5 or 10% CO 2 .
  • Bcl-2 cDNA was obtained in the expression vector pSFFV-Bcl-2 from Dr. Stanley Korsemeyer (Washington University, St. Louis, Mo.; Hockenbery, et al., 1990).
  • Nitric oxide synthase (bNOS) cDNA was obtained from Dr. Solomon Snyder in the vector pNOS (Johns Hopkins University, Baltimore, Md.; Bredt, et al., 1991).
  • p ⁇ MHC 5 .5 CAT containing 5.5 kilobases (Kb) 5' of the mouse ⁇ -myosin heavy chain ( ⁇ MHC) promoter ligated to the chloramphenicol acetyl transferase (CAT) gene, was obtained from Dr. Jeffrey Robbins (University of Cincinnati, College of Medicine, Cincinnati, Ohio; Subramaniam, et al.).
  • p ⁇ MHC 2 .0 CAT containing 2.0 Kb of the rat ⁇ MHC promoter ligated to the CAT gene, was obtained from Dr. Thomas Gustafson (University of Maryland, Baltimore, Md.; Gustafson, et al.).
  • p ⁇ MHC 86 CAT containing 86 base pairs (bp) of the rat ⁇ MHC promoter ligated to the CAT gene, was obtained from Dr. Bruce Markham (Medical College of Wisconsin, Milwaukee, Wis.). The construct was made by 5' truncation of p ⁇ MHC2.0CAT and blunt end ligation to the CAT gene.
  • the sequence of the 86 bp promoter fragment is provided herein as SEQ ID NO:24.
  • pHCA 118 CAT containing 118 bp of the region 5' of the human cardiac ⁇ -actin promoter ligated to the CAT gene, was also obtained from Dr. Larry Kedes (Minty and Kedes).
  • pHSA-150CAT containing 150 bp of the human skeletal muscle ⁇ -actin promoter ligated to the CAT gene, was obtained from Dr. Larry Kedes (University of Southern California, Los Angeles, Calif.; Muscat and Kedes).
  • a construct containing 691 bp (-628 to +63) of the ⁇ -enolase (ENO3) gene was obtained from Dr. Charlotte Peterson (Veterans Administration Medical Center, University of Arkansas, Little Rock, Ark.). A sequence containing this region is presented herein as SEQ ID NO:29.
  • pGL2PV plasmid-gene-1 lght-promoter-vector
  • MCS vector multiple cloning
  • Plasmid pGLHRE (FIGS. 1B, 2A, 3A) was made by blunt-1 lighting the 240 bp HREE1 fragment (SEQ ID NO:6) into the SmaI site of the MCS of pGL2PV (FIG. 1A).
  • Plasmid pGLHSA-150HRE (FIG. 2B) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 150 bp HindIII-SmaI fragment from pHSA-150CAT containing a fragment of the human skeletal actin (HSA) promoter.
  • Plasmid pGL ⁇ MHC 86 HRE (FIG. 2C) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 120 bp HindIII-EcoRI fragment from p ⁇ MHC 86 CAT containing 86 bp (SEQ ID NO:24) of the human ⁇ -myosin heavy chain ( ⁇ -MHC) promoter. The EcoRI end of the 120 bp fragment was filled in with DNA polymerase I using standard methods (Sambrook, et al.) before blunt end ligation to the vector SmaI site.
  • Plasmid DGL ⁇ MHC 86 -GATA-HRE was made by cloning a 36 bp oligonucleotide (SEQ ID NO:1; described above), containing a duplicated GATA 4 box into the HindIII site (filled in with polymerase) of plasmid pGL ⁇ MHC 86 HRE, upstream of the 86 bp promoter fragment.
  • Plasmid pGLHCA 118 HRE (FIG. 2D) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 188 bp HindIII-EcoRI fragment from pHCA 118 CAT, containing 118 bp of the human cardiac actin (HCA) promoter plus 70 bp of actin exon 1.
  • the EcoRI end of the 188 bp fragment was filled in with DNA polymerase I as above before blunt end ligation to the vector SmaI site.
  • Plasmid pGL ⁇ MHC 1 .2 HRE (FIG. 3B) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 1.2 kb HindIII-EcoRI fragment from p ⁇ MHC 20 CAT containing 1.2 kb of the human ⁇ -MHC promoter. The EcoRI end of the 1.2 kb fragment was filled in as above in prior to cloning.
  • Plasmid pGLPKM 460 containing 460 bp of the rat muscle specific pyruvate kinase (PKM) gene promoter and 140 bp of the PKM coding sequence (SEQ ID NO:7), was created using polymerase chain reaction (PCR) as follows. PKM-specific primers containing endonuclease restriction sites near their 5' end were designed based on the nucleotide sequence of the PKM gene (Takenaka, et al., 1989). PKM primer F (SEQ ID NO:2) contained a KpnI site, while PKM primer R (SEQ ID NO:3) contained a XhoI site.
  • PCR polymerase chain reaction
  • PCR was carried out using the above primers and 1 ⁇ g of rat heart genomic DNA as a template for 25 cycles using standard procedures and a Perkin-Elmer (Norwalk, Conn.) DNA thermal cycler.
  • the PCR product (FIG. 6A) was purified by agarose gel electrophoresis, cut with KpnI and XhoI, and cloned into KpnI/XhoI cut pGL2BV (FIG. 6B; Promega Corp., Madison, Wis.), generating pGLPKM 460 (FIG. 6C).
  • Plasmid pGLPKM 285 (FIG. 6E) was generated by digesting pGLPKM 460 with SmaI to drop out the -460 to -285 portion of the promoter, and religating the vector.
  • pGLPKM D (FIG. 6D) was generated by digesting pGLPKM 460 with SmaI to isolate the -460 to -285 portion of the promoter, and cloning that fragment into pGL2PV (Promega Corp.) that had been cut with SmaI.
  • Plasmid pGLET-1 700 (FIG. 7C), containing 700 bp of the human ET-1 gene promoter (SEQ ID NO:8), was created using PCR to amplify HeLa cell genomic DNA as described above. ET-1 specific primers were designed based on the promoter sequence (Inoue, et al., 1989) of the ET-1 gene.
  • the forward primer (SEQ ID NO:4) contained PstI and KpnI sites, while the reverse primer (SEQ ID NO:5) contained HindIII and XbaI sites.
  • the PCR product (FIG. 7A) was purified by gel electrophoresis, cut with KpnI and HindIII, and cloned into KpnI/HindIII cut pGL2BV (FIG. 7B; Promega Corp.).
  • Plasmid pGLENO 628 was constructed by cloning a blunt ended genomic DNA containing an ENO3 promoter fragment (-628 to +63; SEQ ID NO:29), isolated from a lambda gt10 human genomic library, into the SmaI site of pGL2BV.
  • Plasmid p ⁇ MHC 1 .2 HRE-NOS (FIG. 4B) was made by digesting plasmid pGL ⁇ MHC 1 .2 HRE (FIG. 4A) with HindIII and EcoRV to drop out the luciferase cDNA and replacing it with a HindIII/XbaI fragment from pNOS containing a full length NOS CDNA.
  • Plasmid p ⁇ MHC 1 .2 HRE-Bcl-2 (FIG. 5B) was made by digesting pSFFV-Bcl-2 with SalI, blunting the vector as described above, removing the SFFV promoter from the linearized vector with an EcoRI digest, and replacing the SFFV promoter with a SmaI/EcoRI fragment from pgL ⁇ MHC 1 .2 HREE containing the 1.2 kb ⁇ MHC promoter fragment and the 240 bp HREE1.
  • Plasmid p ⁇ MHC 5 .5 HRE-CAT was made by inserting the 240 bp HREE1 immediately 5' of the AMHC promoter of p ⁇ MHC 5 .5 CAT.
  • Adenoviral constructs are made using standard methods (e.g., Friedman,, et al., 1986; Hertz and Gerard, 1993), as follows.
  • Ad ⁇ MHC1.2Bcl2HREE is made by inserting a 3.34 Kb EcoRI/HindIII fragment from p ⁇ MHC1.2-Bcl-2 (containing 1.2 Kb of the ⁇ -MHC promoter, 1.9 Kb Bcl-2 CDNA, and 240 bp HREE1) into pAPLCMV digested with EcoRI and HindIII to drop out the CMV promoter and CAT gene.
  • pAPLCMV which may be obtained from Dr. Larry Kedes (University of Southern California, Los Angeles, Calif.; Kass-Eisler, et al., 1993), is a base replication deficient adenoviral expression vector.
  • the backbone adenoviral vector for recombination, p9M17 may also be obtained from Dr. Larry Kedes.
  • Recombinant pAPLCMV (pAd ⁇ MHC1.2bcl-2HRE) and p9M17 are used to co-transfect 293 cells (ATCC) to propagate the adenovirus.
  • pGLHRE Constructs pGLHRE, pGLHSA-150HRE, p ⁇ MHC 5 .5 HRE-CAT, pGL ⁇ MHC 1 .2 HRE, pGLHCA 118 HRE and pGL-Eno 628 were tested for tissue-specific expression and hypoxia inducibility in HeLa cells, Hep G2 cells, differentiated C2C12 muscle myotubes, and cardiac myocytes.
  • HeLa cells, C2C12 myocytes, and cardiac myocytes were transfected with the indicated plasmid DNA by the standard calcium phosphate procedure (Ausubel, et al.).
  • DNA for transfection was prepared by resuspending an ethanol-precipitated pellet containing 20 ⁇ g of the plasmid DNA in 450 ⁇ l ddH 2 O and adding 50 ⁇ l of 2.5 mM CaCd 2 .
  • the precipitate was added to the dishes containing the cells and the dishes were incubated overnight.
  • the cells were washed twice with 5 ml PBS and fed with 10 ml of complete medium. The cells were then allowed to recover for 24 hours before incubation under an atmosphere of 1.0% O 2 , 5% CO 2 , 94% N 2 for an additional 20 hours.
  • Cells transfected and treated as above were assayed for expression of the luciferase enzyme using a standard reaction protocol (Titus). Briefly, 1 ml of CCLR and 1 ml of LAR were allowed to equilibrate at room temperature. The culture medium in the dish containing the cells to be assayed was removed and the cells were rinsed twice in PBS buffer.
  • Constructs of the present invention were injected directly into cardiac tissue using techniques described in Buttrick, et al., (1992) and Buttrick, et al., (1993). Briefly, adult female Wistar rats were anesthetized with an intraperitoneal injection of chloral hydrate (0.7 ml/100 g of a 4% solution). Cardiac injections were made directly into the apex of the heart through a lateral thoractomy, after which the heart was replaced in the chest, the rats were briefly hyperventilated, and the incision closed.
  • hypoxia-inducibility of vector expression was assayed as follows. Hearts were excised approximately 20 hours after the induced ischemia and the ventricles were washed with ice-cold phosphate buffered saline (PBS). The tissue was suspended in 1 ml of ice-cold PBS containing 20% sucrose and homogenized with a Polytron (Kinematica, Switzerland) for 45 sec. After centrifugation at 10,000 ⁇ g for 10 min supernatants were analyzed for luciferase expression by the assay method described above. Protein was measured using a BioRad assay kit (BioRad Laboratories, Hercules, Calif.).
  • C2C12 myoblasts were cotransfected with pSV2Neo (Minty and Kedes) and a test plasmid at a ratio of 1:19 (1 ⁇ g pSV2Neo+19 ⁇ g test plasmid) using standard methods (Minty and Kedes, 1986).
  • Test plasmids were pGLHRE, pGLHCA 118 HRE, pGL ⁇ MHC 1 .2 HRE, pSFFV-Bcl-2, and pNOS-HRE. Cultures were selected on day 2 following transfection with 400 ⁇ g/ml of the neomycin drug G418 (Gibco/BRL). Colonies of cells resistant to G418 appeared after 10 to 14 days.
  • the resistant cells were pooled. Mass cultures were assayed for the expression of luciferase as described above or by Northern blot assay (Webster, et al., 1993) for the expression of Bcl-2 or NOS RNA. Stable lines were positive for expression of the transfected genes.
  • Mass cultures were subjected to differentiation conditions by transferring them to low mitogen medium (DMEM with 2% horse serum) and were analyzed visually for differentiation into myotubes. There was no apparent difference between transfected and control cells. Approximately 40% of cells were fused into multinucleate myotubes after 24 h in low mitogen medium. All cultures contained approximately 74% myotubes after 48 h.
  • low mitogen medium DMEM with 2% horse serum
  • Reporter-transfected cells differentiated normally and respond to hypoxia with the predicted induction of reporter. NOS-transfected cells appeared normal both during the hypoxia and during subsequent reoxygenation.
  • a stable line of C2C12 cells that constitutively over-expresses Bcl-2 (without HREE1) was also constructed as described above, and the cells showed normal growth and differentiation characteristics.
  • Plasmid pGLPKM 460 was transfected into C2C12 cells and cardiac myocytes and assayed for luciferase activity as described in Example 1.
  • HREKM 285 confers hypoxia-induced expression in C2C12 myotubes and cardiac myocytes similar to that obtained with pGLPKM 460 .
  • This level of hypoxia induction is at least equivalent to that obtained using HREE1 (SEQ ID NO:6).
  • the PKM promoter sequence has no significant homology with the erythropoietin HRE consensus, but does share a consensus sequence (SEQ ID NO:31) with the ENO3 promoter fragment (SEQ ID NO:29).
  • This consensus located approximately 88 bp upstream of the transcription start site of PKM and approximately 70 bp upstream of the transcription start site of ENO3, may represent an important element for conferring enhancement of expression in response to hypoxia.
  • Plasmid pGLET-1 700 was transfected into human arterial endothelial cells as described in Example 1. The expression of pGLET-1 700 in these cells was increased 5 fold by incubation of the cells in a hypoxic atmosphere as described above. No significant induction of pGLET-1 700 was observed in any other cell types tested, including HeLa, C2C12, and cardiac myocytes. Elements contained within the 700 bp sequence have no significant homology with the erythropoietin HRE consensus.
  • the 700 bp fragment of the human ET-1 gene promoter corresponding to the sequence represented herein as SEQ ID NO:8 is effective to (i) restrict expression of genes under its control to the vascular endothelium, and (ii) confer hypoxia-inducibility on the expression of those genes. Accordingly, this fragment, in conjunction with a therapeutic or reporter gene, may be used in the methods of the present invention to both target expression to a selected tissue (vascular endothelium), and confer enhancement of expression by hypoxia.

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Abstract

Methods and compositions relating to chimeric genes containing (i) a tissue-specific promoter and (ii) a hypoxia response enhancer element, both of which are operably linked to a selected gene, such as a reporter gene, therapeutic gene (e.g., bcl-2, NOS, catalase and SOD), or deleterious gene are disclosed. Expression of the selected gene is enhanced in the target tissue under hypoxic conditions, such as conditions encountered during episodes of ischemia and reperfusion. The methods and compositions may be used as therapeutics and/or diagnostics.

Description

This work was supported in part by NIH Grant # 5R29HL44578-04. Accordingly, the United States Government has certain rights in this invention.
FIELD OF THE INVENTION
This invention relates to chimeric genes (e.g., carried on expression vectors) containing therapeutic genes whose expression is under the control of tissue specific and hypoxia response enhancer elements.
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BACKGROUND OF THE INVENTION
Each year, over a half-million Americans die from heart attacks. Even more--close to 700,000--have non-fatal heart attacks. For these surviving victims, a portion of the heart is usually damaged irreparably. Such cell death of cardiac tissue, called myocardial infarction, is due in large part to tissue damage caused by ischemia and/or ischemia followed by reperfusion.
Similar ischemic damage may occur in many other tissues when the blood supply to the tissue is reduced or cut off. Stroke, deep vein thrombosis, pulmonary embolus, and renal failure are examples.
Surviving victims of ischemic episodes, such as heart attacks, are at substantially greater risk for subsequent episodes of ischemia, which in many cases prove debilitating or fatal. Thus, it would be desirable to have therapeutic methods and compositions by which survivors of heart attacks and other types of ischemic insults could lower the risk of tissue damage due to recurrent ischemic/reperfusion episodes.
SUMMARY OF THE INVENTION
In one aspect, the invention includes a method for reducing ischemic injury to a cell exposed to hypoxic conditions. The method includes introducing into the cell a chimeric gene containing a hypoxia response element, a therapeutic gene, and a tissue-specific promoter operably linked to the therapeutic gene to control transcription of the therapeutic gene in the cell, where the element is effective to modulate expression of the therapeutic gene. Exposing the cell to hypoxic conditions enhances expression of the gene and expression of the gene is effective in reducing ischemic injury to the cell. The method may be applied to, for example, cardiac cells using a cardiac-specific promoter, kidney cells using a kidney-specific promoter, brain cells using a brain-specific promoter, and vascular endothelium cells using a vascular endothelium-specific promoter. The hypoxia response element may be selected from, for example, the erythropoietin HRE element (HREE1), muscle pyruvate kinase (PKM) HRE element, β-enolase (enolase 3; ENO3) HRE element and endothelin-1 (ET-1) HRE element. The therapeutic gene may be selected from, for example, nitric oxide synthase (NOS), B-cell leukemia/lymphoma 2 (bcl-2), superoxide dismutase (SOD) and catalase.
In a related aspect, the invention includes a chimeric gene, containing a hypoxia response element, a tissue-specific promoter heterologous to the element, and a therapeutic gene. The promoter is operably linked to the therapeutic gene and the element is effective to modulate expression of the therapeutic gene. The method may be used with a variety of cell types and corresponding promoters, for example, as identified above. Suitable cardiac-specific promoters include the α-MHC5.5 promoter, α-MHC86 promoter, and human cardiac actin promoter. Suitable kidney-specific promoters include the renin promoter. Suitable brain-specific promoters include the aldolase C promoter and the tyrosine hydroxylase promoter. Suitable vascular endothelium-specific promoters include the Et-1 promoter and vonWillebrand factor promoter. Hypoxia response enhancer element useful with the method include HREE1, PKM HRE element, ENO3 HRE element and ET-1 HRE element. Therapeutic genes useful with the method include NOS, Bcl-2, SOD and catalase. The chimeric gene may also be contained in an expression vector.
Another aspect of the present invention includes the above-described chimeric gene carried in an expression vector. The expression vector may be a plasmid, adenovirus vector, retrovirus vector, or the like.
In still another aspect, the invention includes a chimeric gene which contains a hypoxia response element, a tissue-specific promoter heterologous to the element, and a deleterious gene. The promoter is operably linked to the deleterious gene, and the element is effective to modulate expression of the deleterious gene. Suitable promoters include tumor-specific promoters, such as alpha fetoprotein (AFP) promoter. Suitable hypoxia response elements are as articulated above. Deleterious genes useful in this aspect include a viral thymidine kinase gene (tk), such as the herpes simplex virus (HSV) tk.
In a related aspect, the invention includes a method of causing injury to a cell exposed to hypoxic conditions. The method includes introducing into the cell a vector containing a hypoxia response element, a deleterious gene, and a tissue-specific promoter operably linked to the gene and capable of controlling transcription of the gene in the cell. Exposing the cell to hypoxic conditions enhances expression of the gene, and expression of the gene is effective to cause injury to the cell. Promoters useful with this method include tumor-specific promoters such as the AFP promoter. Specific hypoxia response elements and deleterious genes useful with the method are also as identified above.
The invention further includes a substantially isolated polynucleotide having a sequence corresponding to hypoxia response enhancer element(s) (HREE(s)) present in a control region of the muscle pyruvate kinase gene. The element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region. In a related aspect, the invention includes an HRE element derived from a muscle pyruvate kinase gene.
Also included in the invention is a substantially isolated polynucleotide having a sequence corresponding to hypoxia response element(s) present in a control region of the endothelin-1 gene. The element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region. In a related aspect, the invention includes an HRE element derived from an endothelin-1 gene.
Another aspect of the invention includes a substantially isolated polynucleotide having a sequence corresponding to hypoxia response element(s) present in a control region of the enolase 3 (ENO3) gene. The element may be derived from the promoter region, 5' untranslated region, or 3' untranslated region. In a related aspect, the invention includes an HRE element derived from an ENO3 gene.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B show a schematic diagram of the construction of plasmid pGLHRE (FIG. 1B) from plasmid pGL2PV (FIG. 1A).
FIGS. 2A, 2B, 2C and 2D show a schematic diagram of the construction of plasmids pGLHSA-150HRE (FIG. 2B), pGLαMHC86 -HRE (FIG. 2C), and pGLHCA118 HRE (FIG. 2D), from plasmid pGLHRE (FIG. 2A).
FIGS. 3A and 3B show a schematic diagram of the construction of plasmid pGLαMHC1.2 HRE (FIG. 3B) from plasmid pGLHRE (FIG. 3A).
FIGS. 4A and 4B show a schematic diagram of the construction of plasmid pGLαMHC1.2 HRE-NOS (FIG. 4B) from plasmid pGLαMHC1.2 HRE (FIG. 4A).
FIGS. 5A and 5B show a schematic diagram of the construction of plasmid pαMHC1.2 -HRE-Bcl-2 (FIG. 5B) from plasmid pSFFV-Bcl-2 (FIG. 5A).
FIGS. 6A, 6B, 6C, 6D and 6E show a schematic diagram of the construction of plasmids pGLPKM460 (FIG. 6C), pGLPKMD (FIG. 6D), and pGLPKM285 (FIG. 6E) from plasmid pGL2BV (FIG. 6B) and a fragment of the PKM promoter (FIG. 6A; SEQ ID NO:7).
FIGS. 7A, 7B and 7C show a schematic diagram of the construction of plasmid pGLET-1700 (FIG. 7C) from plasmid pGL2BV (FIG. 7B) and a fragment of the ET-1 promoter (FIG. 7A; SEQ ID NO:8).
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 is the sense strand nucleotide sequence of a GATA4 enhancer element (Molkentin, et al., 1984).
SEQ ID NO:2 is the nucleotide sequence of muscle pyruvate kinase (PKM) sense strand primer F.
SEQ ID NO:3 is the nucleotide sequence of PKM reverse strand primer R.
SEQ ID NO:4 is the nucleotide sequence of endothelin-1 (Et-1) sense strand primer F.
SEQ ID NO:5 is the nucleotide sequence of Et-1 reverse strand primer R.
SEQ ID NO:6 is the nucleotide sequence of hypoxia response enhancer element 1 (HREE1), derived from the erythropoietin (EPO) gene (Semenza and Wang), and containing 4 tandem copies of a hypoxia response enhancer (HRE) sequence and cloning linkers.
SEQ ID NO:7 is the nucleotide sequence of a rat muscle pyruvate kinase (PKM) promoter region (Takenaka, et al.).
SEQ ID NO:8 is the nucleotide sequence of a human Et-1 promoter region (Inoue, et al.).
SEQ ID NO:9 is the nucleotide sequence of a human cardiac actin promoter region (Minty and Kedes).
SEQ ID NO:10 is a nucleotide sequence containing a portion of the rat cardiac α-myosin heavy chain promoter region (Mahdavi, et al.; GenBank Accession # K01464).
SEQ ID NO:11 is a nucleotide sequence containing a portion of the mouse cardiac α-myosin heavy chain promoter region (Gulick, J., et al.; GenBank Accession # M62404).
SEQ ID NO:12 is the nucleotide sequence of a human B-cell leukemia/lymphoma 2 (bcl-2) gene (Tsujimoto, et al.; GenBank Accession # M13994).
SEQ ID NO:13 is the predicted amino acid sequence from SEQ ID NO:12.
SEQ ID NO:14 is the nucleotide sequence of a rat nitric oxide synthase (bNOS) gene (Bredtet al.; EMBL Accession # X59949).
SEQ ID NO:15 is the predicted amino acid sequence from SEQ ID NO:14.
SEQ ID NO:16 is the nucleotide sequence of a human bcl-2 fusion gene (Seto, et al.; EMBL Accession # X06487).
SEQ ID NO:17 is the predicted amino acid sequence from SEQ ID NO:16.
SEQ ID NO:18 is the nucleotide sequence of a human NOS-1 gene (Fujisawa, et al.); DDBJ Accession # D16408; NCBI Seq ID 506339)
SEQ ID NO:19 is the predicted amino acid sequence from SEQ ID NO:18.
SEQ ID NO:20 is the nucleotide sequence of a human NOS-SN gene (Nakane, et al.; GenBank Accession # L02881)
SEQ ID NO:21 is the predicted amino acid sequence from SEQ ID NO:20.
SEQ ID NO:22 is the nucleotide sequence of a 256 base pair (bp) 3' EPO-1 hypoxia response enhancer element (Semenza and Wang).
SEQ ID NO:23 is the nucleotide sequence of a 42 bp 3' EPO-1 hypoxia response enhancer element (Madan, et al.).
SEQ ID NO:24 is the nucleotide sequence of an 86 bp rat αMHC promoter region.
SEQ ID NO:25 is the nucleotide sequence of a mouse catalase gene (Reimer, et al.; GenBank # L25069).
SEQ ID NO:26 is the predicted amino acid sequence from SEQ ID NO:25.
SEQ ID NO:27 is the nucleotide sequence of a human manganese superoxide dismutase (SOD) gene (Clair, et al.; EMBL # X59445).
SEQ ID NO:28 is the predicted amino acid sequence from SEQ ID NO:27.
SEQ ID NO:29 is the nucleotide sequence of a human β-enolase (ENO3) gene (Giallongo, et al.; EMBL # X56832) between nucleotides -628 to +63.
SEQ ID NO:30 is the predicted amino acid sequence from SEQ ID NO:29.
SEQ ID NO:31 is a consensus sequence of a region present in both the PKM and ENO3 promoters.
DETAILED DESCRIPTION OF THE INVENTION Definitions
"Ischemia" is defined as an insufficient supply of blood to a specific organ or tissue. A consequence of decreased blood supply is an inadequate supply of oxygen to the organ or tissue (hypoxia). Prolonged hypoxia may result in injury to the affected organ or tissue. "Anoxia" refers to a virtually complete absence of oxygen in the organ or tissue, which, if prolonged, may result in death of the organ or tissue.
"Hypoxic condition" is defined as a condition under which a particular organ or tissue receives an inadequate supply of oxygen.
"Anoxic condition" refers to a condition under which the supply of oxygen to a particular organ or tissue is cut off.
"Reperfusion" refers to the resumption of blood flow in a tissue following a period of ischemia.
"Ischemic injury" refers to cellular and/or molecular damage to an organ or tissue as a result of a period of ischemia and/or ischemia followed by reperfusion.
An "element", when used in the context of nucleic acid constructs, refers to a region of the construct or a nucleic acid fragment having a defined function. For example, a hypoxia response enhancer element is a region of DNA that, when associated with a gene operably linked to a promoter, enhances the transcription of that gene under hypoxic conditions.
The term "operably linked", as used herein, denotes a relationship between a regulatory region (typically a promoter element, but may include an enhancer element) and the coding region of a gene, whereby the transcription of the coding region is under the control of the regulatory region.
Two nucleic acid elements are said to be "heterologous" if the elements are derived from two different genes, or alternatively, two different species. For example, a hypoxia response enhancer element from a human erythropoietin gene is heterologous to a promoter from a human myosin gene. Similarly, a hypoxia response enhancer element from a human erythropoietin gene, for example, is heterologous to a promoter from a mouse erythropoietin gene.
"Control region" refers to specific sequences at the 5' and 3' ends of eukaryotic genes which may be involved in the control of either transcription or translation. For example, most eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription initiation site. Similarly, most eukaryotic genes have a CXCAAT region (X may be any nucleotide) 70 to 80 bases upstream from the start of transcription. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may be the signal for addition of the polyadenylation tail to the 3' end of the transcribed mRNA.
"Chimeric gene" also termed "chimeric DNA construct" refers to a polynucleotide containing heterologous DNA sequences, such as promoter and enhancer elements operably linked to a therapeutic gene. For example, a construct containing a human α-myosin heavy chain (α-MHC) promoter fragment operably linked to a human bcl-2 gene and containing a human erythropoietin gene hypoxia response element comprises an exemplary chimeric gene.
I. Overview of the Invention
The present invention relates to chimeric genes having at least three functional elements: (i) a therapeutic gene, (ii) a tissue-specific promoter, and (iii) a hypoxia response enhancer (HRE) element. The tissue-specific promoter in combination with the HRE element directs expression of the therapeutic gene in a selected tissue under hypoxic conditions.
The gene is preferably introduced into a target tissue as part of a complete expression vector in a pharmaceutically-acceptable vehicle, either by direct administration to the target tissue (e.g., injection into the target tissue), or by systemic administration (e.g., intravenous injection). In the latter case, the gene may be targeted to a selected tissue, for example, by incorporating it in a virion expressing a modified envelope protein designed to bind to receptors preferentially expressed on cells from the selected, or targeted, tissue. Regardless of the delivery means, expression of the gene in tissues other than the target tissue, and under conditions other than hypoxic or anoxic is preferably minimal.
As described below, a variety of therapeutic genes, promoters, HRE elements and gene delivery means may be employed in the practice of the present invention.
II. Tissue Specific Promoters
A promoter, in the context of the present specification, refers to a polynucleotide element capable of regulating the transcription of a gene adjacent and downstream (3') of the promoter. The promoter may contain all of, or only a portion of, the complete 5' regulatory sequences of the gene from which it is derived. A sequence in the promoter region is typically recognized by RNA polymerase molecules that start RNA synthesis.
A promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular tissue. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-specific factor), under certain conditions (e.g., hypoxia, or the presence of an enhancer element in the chimeric gene containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult).
Promoters useful in the practice of the present invention are preferably tissue-specific--that is, they are capable of driving transcription of a gene in one tissue while remaining largely "silent" in other tissue types. It will be understood, however, that tissue-specific promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue). In this regard, a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 5. Preferably, the selectivity ratio is greater than about 15.
It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific", and are contemplated for use with the present invention. For example, promoters that are active in a variety of central nervous system (CNS) neurons may be therapeutically useful in protecting against damage due to stroke, which may effect any of a number of different regions of the brain.
Tissue-specific promoters may be derived, for example, from promoter regions of genes that are differentially expressed in different tissues. For example, a variety of promoters have been identified which are suitable for upregulating expression in cardiac tissue. Included are the cardiac α-myosin heavy chain (AMHC) promoter and the cardiac α-actin promoter.
A further desirable characteristic of promoters useful in the present invention is that they possess a relatively low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissues. One means of achieving this is to select promoters of genes encoding proteins that have a relatively low turnover rate in adult tissue, such as the actin and α-MHC promoters described herein. Another means is to use "silencer" elements, which suppress the activity of a selected promoter in the absence of hypoxia.
The level of expression of a gene under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis). Vectors used for such experiments typically contains a reporter gene, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities.
This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring hypoxia sensitivity.
A number of tissue specific promoters, described below, may be particularly advantageous in practicing the present invention. In most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector.
Alternatively, promoter fragments may be isolated using the polymerase chain reaction (PCR; Mullis, Mullis, et al.). Cloning of amplified fragments may be facilitated by incorporating restriction sites at the 5' ends of the primers.
Promoters suitable for cardiac-specific expression include the promoter from the murine cardiac α-myosin heavy chain gene. The gene contains a 5.5 kbp promoter region which may be obtained as a 5.5 kbp SacI/Sall fragment from the murine αMHC gene (Subramaniam, et al., 1991). Reporter gene constructs utilizing this 5.5 kbp αMHC promoter are expressed at relatively high levels selectively in cardiac tissue (whether or not an HREE is present) and, when present in transgenic animals, are regulated in a similar fashion to the endogenous gene (Subramaniam, et al., 1991).
A smaller fragment of the rat α-MHC promoter may be obtained as a 1.2 kbp EcoRI/HindIII fragment (Gustafson, et al.). As shown in Example 1 and Table 1, below, constructs utilizing the 1.2 kbp rat αMHC promoter are expressed at a low level in the absence of an HREE, and at an intermediate level in the presence of an HREE. These results indicate that the αMHC1.2 promoter is an exemplary promoter to target expression of chimeric genes of the present invention to cardiac tissue. Expression of genes under the control of this promoter fragment is very low in cardiac cells under normal oxygenation conditions, but is increased by about a factor of four under hypoxic conditions when the construct contains HREE1. Expression in cells other than cardiac cells is at background levels.
An 86 bp fragment of the rat αMHC promoter, presented herein as SEQ ID NO:24, restricts expression of reporter genes to cardiac and skeletal muscle (i.e., it has lost some tissue selectivity). Additional cardiac specificity may be conferred to the fragment by ligating (e.g., blunt end ligating) a 36-mer oligonucleotide (SEQ ID NO:1) containing cardiac-specific GATA4 enhancer elements just upstream of base pair -86 (Molkentin, et al., 1984). This promoter fragment also results in low levels of expression in the absence of additional enhancers such as HRE elements. The low level of basal expression induced by the 86 bp fragment, and the ability to upregulate this basal level of expression with a hypoxia response enhancer element are useful properties for a promoter for use with the present invention.
The sequences of exemplary cardiac-specific promoter regions from the rat and mouse AMHC genes are presented herein as SEQ ID NO:10 and SEQ ID NO:11, respectively. Both sequences end just upstream of the ATG initiation codons of their respective genes. Other cardiac-specific promoters include the cardiac α-actin promoter and the myosin light chain-2 (MLC-2) promoter. Constructs described herein utilizing a 118 bp fragment (SEQ ID NO:9) from the human cardiac α-actin (HCA) promoter result in a relatively low level of cardiac-specific expression, which may be increased by the inclusion of an HREE in the expression construct (Example 1, Table 1). The cardiac-specific myosin light chain-2 promoter may be obtained as a 2.1 kbp KpnI/EcoRI fragment from the rat cardiac myosin light chain-2 (MLC-2) gene (Franz, et al.).
Prostate-specific promoters include the 5'-flanking regions of the human glandular kallikrein-1 (hKLK2) gene and the prostate-specific antigen (hKLK3; PSA) gene (Murtha, et al.; Luke, et al.). The renin promoter is suitable for directing kidney-specific expression (Fukamizu, et al.), while the aldolase-C promoter (Vibert, et al.) or the tyrosine hydroxylase promoter (Sasaoka, et al.) may be used to direct expression in the brain. Promoters specific for vascular endothelium cells include the Et-1 promoter (Inoue, et al.) and vonWillebrand factor (Jahrondi and Lynch) promoter.
Tumor-specific promoters include the α-fetoprotein (AFP) promoter, contained in a 7.6 kbp fragment of 5'-flanking DNA from the mouse AFP gene (Marci, et al.). This promoter normally directs expression of the AFP gene in fetal liver and is transcriptionally silent in adult tissues. However, it can be abnormally reactivated in hepatocellular carcinoma (HCC), conferring tumor-specific expression in adult tissue (Marci, et al.).
The above promoters are exemplary promoters for use with the present invention. Other promoters suitable for use with the present invention may be selected by one of ordinary skill in the art following the guidance of the present specification.
III. Hypoxia Response Enhancer Elements
Therapeutic genes contained in constructs of the present invention are preferably expressed at low levels, if at all, under conditions of normal oxygenation (minimizing any side effects). Under conditions of hypoxia, however, expression of the genes is increased, affording protection to the target tissue. The elevated expression under hypoxic conditions is conferred by the presence of one or more hypoxia response enhancer (HRE) elements.
HRE elements contain polynucleotide sequences that may be located either upstream (5') or downstream (3') of the promoter and/or therapeutic gene. The HRE element (HREE) is typically a cis-acting element, usually about 10-300 bp in length, that acts on a promoter to increase the transcription of a gene under the control of the promoter. Preferably, the promoter and enhancer elements are selected such that expression of a gene regulated by those elements is minimal in the presence of a healthy supply of oxygen, and is upregulated under hypoxic or anoxic conditions.
Hypoxia response enhancer elements are found in association with a number of genes, including the erythropoietin (EPO) gene. Exemplary HRE elements from the EPO gene are presented herein as SEQ ID NO:6, SEQ ID NO:22 and SEQ ID NO:23. The element having the sequence represented as SEQ ID NO:22 results in approximately a five-fold induction of reporter gene expression under hypoxic conditions (Semenza and Wang), while, the element having the sequence represented as SEQ ID NO:23 results in approximately a 17-fold increase in activity under hypoxic conditions (Madan, et al.)
Experiments performed in support of the present invention (e.g., Example 1) demonstrate that expression of constructs containing HREE1 (SEQ ID NO:6) is increased by approximately 5- to 7-fold in response to hypoxic conditions. These results indicate that the HREE1 element is fully functional when fused to muscle and cardiac specific promoters and that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters.
Expression of constructs containing a fragment (SEQ ID NO:29) from the control region of the enolase 3 (ENO3) gene was induced approximately 5 to 8 fold by hypoxia in C2C12 cells and cardiac myocytes respectively (see Table 1). These results suggest that the HREE in the ENO3 promoter fragment may be a particularly effective HREE for hypoxia induction in constructs containing a tissue-specific promoter, such as a cardiac or skeletal muscle promoter.
According to the present invention, exemplary hypoxia response enhancer elements may also be isolated from regulatory regions of both the muscle glycolytic enzyme pyruvate kinase (PKM) gene (Takenaka, et al.), the human muscle-specific β-enolase gene (ENO3; Peshavaria and Day), and the endothelin-1 (ET-1) gene (Inoue, et al.). The HRE regions from the PKM gene and the ET-1 gene, identified in experiments performed in support of the present invention (see Materials and Methods, Examples 4 and 5), are presented herein as SEQ ID NO:7 and SEQ ID NO:8, respectively.
Example 4 demonstrates that the expression of pGLPKM, a plasmid containing the HRE element from the PKM gene, in transfected C2C12 myotubes and neonatal cardiac myocytes was increased by 6±2 (n=4) fold in both cell types by incubation of the cells in a hypoxic atmosphere. A portion of this HRE element, obtained by digesting with SmaI to cut at an internal SmaI site, localized the hypoxia response sequence to a 200 bp fragment. This fragment, termed HREPKM285, confers hypoxia-induced expression in C2C12 myotubes and cardiac myocytes that is at least equivalent to that obtained using HREE1 (SEQ ID NO:6).
Both PKM and ENO3 promoters contain a common sequence element (SEQ ID NO:31) located at 5' -88 and -70 bp respectively from the transcription start sites. An oligonucleotide containing this sequence may be sufficient to confer hypoxia response characteristics to constructs of the present invention.
Data presented in Example 5 show that expression of pGLET-1700, containing 700 bp of the human ET-1 gene promoter (SEQ ID NO:8), in transfected human arterial endothelial cells was increased approximately 5 -fold by incubation of the cells in a hypoxic atmosphere. No hypoxia-induced increase in pGLET-1700 expression was seen in other cell types, including HeLa cells, C2C12 cells, and cardiac myocytes. Accordingly, the 700 bp fragment may be used to target hypoxia regulated genes specifically to cells of the vascular endothelium, since the fragment contains element(s) conferring tissue specificity (i.e., elements effective to target expression exclusively to the vascular endothelium), as well as HRE element(s) effective to upregulate transcription of a gene under control of the fragment during hypoxic conditions.
It will be appreciated that the present invention includes the use of HRE elements not explicitly identified above. Additional HRE elements may be identified, for example, as detailed in Examples 4 and 5. Further, promoter deletion and mutation analyses (e.g., as described above and in Webster and Kedes) may be used to identify such elements in other hypoxia responsive genes. A number of such responsive target genes have been shown to be induced when cells are exposed to hypoxia in vitro (e.g., Heakock and Sutherland).
It will also be appreciated that, in certain circumstances, the tissue-specific promoter and the hypoxia response enhancer element(s) of the present invention may be derived from a contiguous polynucleotide sequence from a single gene (e.g., as shown above for the ET-1 promoter region, which contains HRE element(s) and also imparts endothelial cell-specific expression).
IV. Therapeutic Genes
The present invention may be used to alleviate a number of disease conditions resulting from hypoxic and/or anoxic conditions due to ischemia where cell and tissue damage results from ischemia and ischemia followed by reperfusion. The invention is particularly suitable in cases where the subject is diagnosed to be at risk for an ischemic episode in a particular tissue.
For example, it is recognized that virtually all surviving heart attack victims are at significantly increased risk for recurrent episodes of myocardial ischemia. Such subjects would benefit from the introduction of constructs capable of expressing therapeutic genes into their cardiac tissue in order to decrease the risk of injury to the tissue during any subsequent ischemic episodes. Such constructs may serve to protect, for example, cardiac and vascular endothelial tissues from ischemic damage and thereby prevent the progression of the heart disease.
Recurrent ischemia and reperfusion typically results in oxidative damage to cells from reactive oxygen species (free radicals), such as peroxides, that are generated during redox switching (Frei). Contact of fresh blood with damaged or dead cells induces the influx of neutrophils, or pus cells, which kill heart cells which would otherwise have recovered. Much of the damage caused by neutrophils has been attributed to superoxide ions. The superoxide anion can damage tissue in several ways. The interaction of the superoxide anion with hydrogen peroxide leads to the production of hydroxyl radicals which are potentially toxic and react rapidly with most organic molecules. Lipids, proteins, and nucleic acids may all be primary targets for such oxidative damage. The extent and type of damage depend on the severity and nature of the hypoxic stress. For example, the stress may cause cellular damage, initiating an inflammatory response with neutrophil attack and subsequent tissue necrosis. Alternatively, the stress may initiate apoptosis (programmed cell death) to eliminate the damaged cells.
Regardless of the mechanism by which tissue death occurs (necrosis or apoptosis), the damage caused by ischemia-reperfusion episodes is typically the result of redox reactions and is quantitatively related to the severity and duration of the ischemia. For example, in the case of the myocardium, a severe heart attack may result in extensive damage (e.g., infarction of 30% to 40% of the left ventricle), whereas moderate angina and silent repetitive ischemia may result in relatively minor damage during each episode.
While the pathology of ischemia in tissues is complex, resulting in multiple potential targets for therapeutic intervention, several classes of targets are particularly suitable for therapeutic intervention in accordance with the teachings of the present invention. These include anti-oxidant systems, that may intervene immediately at the sites of intracellular redox reactions to minimize damage, and vasodilator systems, that may minimize the severity of the ischemia by increasing blood flow to vulnerable tissues. Antioxidant proteins amenable for use with the present invention include gene products of Bcl-2, catalase and superoxide dismutase (SOD) genes, while proteins with vasodilative properties include nitric oxide synthase (NOS), which produces the vasodilator nitric oxide (NO).
Bcl-2, an integral inner mitochondrial membrane protein of relative molecular mass ˜25 kDa, has been shown to protect certain cells against apoptosis (Hockenbery, et al., 1990) by acting as an antioxidant (Hockenbery, et al., 1993). Bcl-2 may be an effective therapeutic gene for reducing damage to tissues during ischemic episodes because apoptosis may be a common response of many tissues, including the heart, to oxidative stress (Williams and Smith; Gottlieb, et al.
The enzyme superoxide dismutase (SOD) catalyzes the decomposition of the superoxide anion to peroxide. Enzymes such as superoxide dismutase, free radical scavengers or agents which prevent the influx on neutrophils are able to increase the salvage of heart muscle cells. The enzyme catalase in turn catalyzes the conversion of peroxides to water. Exemplary sequences of a SOD gene and a catalase gene are presented herein as SEQ ID NO:27 and SEQ ID NO:25, respectively. The sequence presented herein as SEQ ID NO:27 encodes a manganese SOD, which has a relatively long half-life. A related sequence, of a human Cu/Zn SOD, may be found in Gorechi, et al. The Cu/Zn SOD has a shorter half-life than the manganese SOD.
Endothelial-derived nitric oxide (NO) regulates the expression of vasoconstrictors and growth factors by the vascular endothelium (Kourembanas, et al.). Under hypoxia, endothelial cells typically increase expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor. This increase in expression can be reduced or prevented by exposure to NO (Kourembanas, et al.). One of the effects of ET-1 induced vasoconstriction is decreased blood flow to the affected organ or tissue, which can exasperate hypoxic damage due to ischemia. According to the present invention, such damage may be reduced by providing NO to the affected tissue through the expression of a NOS gene under the control of a vascular epithelium or cardiac-specific promoter and hypoxia response enhancer element.
Therapeutic genes of the present invention may be preferably derived from the same or related species as the one to which the methods and compositions of the present invention are applied. For example, for therapeutic treatment of a dog, it may be desirable to utilize a construct containing a therapeutic gene cloned from a dog. Similarly, for treatment of human conditions, it may be desirable to utilize therapeutic genes cloned from human-derived nucleic acids.
The genes encoding the proteins discussed above represent exemplary therapeutic genes useful in the practice of the present invention. It will be appreciated, however, that following the teachings and guidance of the present specification, one of skill in the art may select other therapeutic genes effective to reduce cellular damage due to hypoxia or ischemia, and that the use of such genes is considered to be within the scope of the present invention.
V. Deleterious Genes
In another aspect, the present invention includes constructs containing deleterious genes, rather than therapeutic genes. Expression of the deleterious genes is targeted to tissues which are harmful (e.g., malignant tumors) or otherwise undesirable. Promoters and hypoxia response elements may be selected as described above. Promoters useful in this aspect of the invention preferably restrict expression only to the undesirable tissue. For example, as discussed above, the AFP promoter can be activated in hepatocellular carcinoma (HCC), conferring tumor-specific expression in adult tissues (Marci, et al.).
Deleterious genes include a viral thymidine kinase gene (tk), such as the herpes simplex virus (HSV) tk. This gene is not deleterious by itself, but when expressed, viral TK can phosphorylate ganciclovir (GCV), turning GCV into a cytotoxic compound. Since tumor cells are typically hypoxic, constructs having a tumor-specific promoter operably linked to a viral tk and an HREE may be used in conjunction with GCV to selectively kill tumor cells.
VI. Expression Vectors
Chimeric genes of the present invention are preferably incorporated into expression vectors capable of expressing a therapeutic gene product in a selected eukaryotic host cell (i.e., a target tissue). Such expression vectors may contain, in addition to the chimeric gene, various other sequences useful for effective expression of the therapeutic gene in selected tissues. Such sequences may include, for example, sequences necessary for the termination of transcription. These sequences are transcribed as polyadenylated segments in the untranslated portion of the MRNA encoding the desired therapeutic protein. The 3' untranslated regions may also include transcription termination sites.
Molecular techniques and methods useful in the construction of expression vectors are well known in the art (e.g., Ausubel, et al., Sambrook, et al.). Vector constructs made in support of the present invention are designed to express either a reporter gene (e.g., luciferase), or therapeutic genes (e.g., Bcl-2 or NOS). Therapeutic gene expression is under the control of either a ubiquitous promoter (e.g., SV40), or a tissue-specific promoter (e.g., striated muscle or cardiac-specific promoter). Further regulation of expression by hypoxia or anoxia is provided by inclusion of hypoxia response enhancer (HRE) elements (e.g., from the erythropoietin (EPO) gene, muscle specific pyruvate kinase (PKM) gene, enolase 3 (ENO3) gene or the endothelial cell endothelin-1 (Et-1) gene).
The generation of exemplary constructs is described in the Materials and Methods section, below. The results of in vitro experiments to assess the performance of constructs having HREE1 and tissue specific promoters are presented in Example 1 and Table 1. The relative amount of gene expression was measured using a reporter gene (luciferase) in place of a therapeutic gene.
The data shown in Table 1 demonstrate that cells containing constructs having a hypoxia response enhancer element, such as HREE1, in combination with a compatible promoter, express the reporter at levels that are 5 to 7 times greater under hypoxic conditions than under aerobic conditions, and that HREE1 is equally active in different cells and independent of the promoter. The data also demonstrate that expression of constructs containing α-MHC promoters is cardiac specific, and that the basal (aerobic) expression from α-MHC1.2 and HCA promoters is relatively low. Further, the data indicate that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters.
In vivo experiments conducted with plasmids pGLHRE and pGLHCA118 HRE (Example 2, Table 2) demonstrate that gene expression in hearts of rats injected with the plasmids and subjected to ischemia was approximately 2-fold higher than expression in hearts from control animals (not subjected to ischemia). These results indicate that the direct injection of therapeutic constructs of the present invention into cardiac tissue in vivo is effective to result in the expression of genes carried on those plasmids. Further, these results indicate that expression vectors carrying chimeric genes of the present invention are effective to result in significantly increased levels of expression in response to hypoxia caused by ischemia in vivo.
Since expression was measured at 20 hours after a brief (20 minute) episode of ischemia, it will be appreciated that (i) hypoxia-induced expression may peak significantly earlier than 20 hours, and (ii) repeat ischemic episodes may upregulate expression more the single experimental episode used herein. Accordingly, the 2-fold induction may be an underestimate of the level of enhancement of transcription/expression caused by ischemia.
While the experiments described above were performed with cardiac tissue, it will be appreciated that one of ordinary skill in the art having the benefit of the present specification may perform similar manipulations with other tissues subject to ischemic and or ischemic/reperfusion injury, and that such procedures are within the scope of the present invention.
In vitro experiments (Example 3) demonstrate that cells transfected with reporter (pGLHRE, pGLHCA118 HRE, pGLαMHC1.2 HRE) and therapeutic (pSFFV-Bcl-2 and pNOS-HRE) constructs appear normal and respond to stimuli as expected. Reporter-transfected cells differentiate normally and respond to hypoxia with the predicted induction of reporter, while NOS and bcl-2-transfected cells appear normal both during the hypoxia and during subsequent reoxygenation. These results suggest that inclusion of HRE elements, Bcl-2 over-expression, and hypoxia-induced over-expression of NOS is not toxic or deleterious to muscle cells in vitro.
These results also suggest that expression vectors carrying therapeutic genes of the present invention may be effective to protect tissues from ischemic damage. Such protective effects may be assayed in an animal model by, for example, infecting myocardial tissue with an expression vector containing a chimeric gene of the present invention, such as an adenoviral vector expressing a therapeutic gene (e.g., Bcl-2 or SOD), a cardiac-specific promoter, and an HRE element, as described, for instance, in Example 2.
Following infection, the animals may be subjected to repeat ischemic episodes (e.g., 30 minutes to 1 hour) followed by reperfusion (e.g., 1 to 8 hours). Following the last reperfusion, the animals may be sacrificed and the ischemic regions of the myocardium may be tested for the presence and extent of infarction as described, for example, by Thornton, et al., and for the presence of apoptosis as described, for example, in Gottlieb, et al. Sample biopsies may also be assayed for expression of the therapeutic gene by Northern blots.
Similar experiments may be performed using constructs direct (e.g., via an appropriate promoter) to other tissues, such as brain, kidney and vascular endothelium.
VII. Delivery of Constructs to Cells and Tissues
Any of a variety of methods known to those skilled in the art may be used to introduce chimeric genes of the present invention into selected target tissue cells. For example, gene therapy of cardiac tissue has included lipofection, retrovirus and adenovirus-mediated gene transfer, and injection of naked DNA directly into the vascular endothelium or cardiac tissue (Nabel, et al.; Lin, et al.; Leclere, et al.; Flugelman, et al.). These and other methods are discussed more fully in the sections below.
Viral-Mediated Gene Transfer.
Host cells may be transfected with chimeric genes of the present invention by infection with mature virions containing hybrid vectors (the chimeric genes along with selected viral sequences). The virions used to transfect host cells are preferably replication-defective, such that the virus is not able to replicate in the host cells.
The virions may be produced by co-infection of cultured host cells with a helper virus. Following coinfection, the virions are isolated (e.g., by cesium chloride centrifugation) and any remaining helper virus is inactivated (e.g., by heating). The resulting mature virions contain a chimeric gene of the present invention and may be used to infect host cells in the absence of helper virus. Alternatively, high titers of replication-defective recombinant virus, free of helper virus, may be produced in packaging cell lines containing those components for which the virus is defective (Miller).
Several types of viruses, including retroviruses, adeno-associated virus (AAV), herpes virus, vaccinia virus, and several RNA viruses may be amenable for use as vectors with chimeric gene constructs of the present invention. Each type of virus has specific advantages and disadvantages, which are appreciated by those of skill in the art. Methods for manipulating viral vectors are also known in the art (e.g., Grunhaus and Horowitz; Hertz and Gerard; and Rosenfeld, et al.)
Retroviruses, like adeno-associated viruses, stably integrate their DNA into the chromosomal DNA of the target cell. Unlike adeno-associated viruses, however, retroviruses typically require replication of the target cells in order for proviral integration to occur. Accordingly, successful gene transfer with retroviral vectors depends on the ability to at least transiently induce proliferation of the target cells.
Retroviral vectors are attractive in part due to the efficiency of transfection--some vectors can stably transduce close to 100% of target cells. The use of retroviral vectors for in vivo gene therapy has been limited, in part, by the requirement of appropriate viral receptors on the target cell. Because the identities of most retroviral receptors are unknown, it has not been possible to determine the distribution of receptors in different cell types. Accordingly, the targeting of specific cell types by retroviral vectors has in many cases proven problematic.
This difficulty may be circumvented by modifying the envelope protein of the retrovirus to contain a ligand for a known endogenous (not necessarily viral) receptor expressed on the target cells. An application of this technique is described in detail by Kasahara. Preferably, the virus also contains an unmodified envelope protein to facilitate cell entry. A number of receptors, such as desmin, E-selectin, and A-CAM, are expressed preferentially on cardiac cells and may be amenable to this approach (e.g., Hansen and Stawaski; Lefer, et al.; Youker, et al.).
Adeno-associated viruses are capable of efficiently infecting nondividing cells and expressing large amounts of gene product. Furthermore, the virus particle is relatively stable and amenable to purification and concentration. Replication-defective adenoviruses lacking portions of the E1 region of the viral genome may be propagated by growth in cells engineered to express the E1 genes (Jones and Shenk; Berkner; Graham and Prevea). Most of the currently-used adenovirus vectors carry deletions in the E1A-E1B and E3 regions of the viral genome. A number of preclinical studies using adenoviral vectors have demonstrated that the vectors are efficient at transforming significant fractions of cells in vivo, and that vector-mediated gene expression can persist for significant periods of time (Rosenfeld, et al.; Quantin, et al.; Stratford-Perricaudet, et al., 1992a; Rosenfeld, et al.; L. D. Stratford-Perricaudet, et al., 1992b; Jaffe, et al.). Several studies describe the effectiveness of adenovirus-mediated gene transfer to cardiac myocytes (Kass-Eisler, et al.; Kirshenbaum, et al.).
Herpes virus vectors (Breakefield and DeLuca; Freese, et al.) are particularly well suited for the delivery and expression of foreign DNA in cells of the central nervous system (CNS), since they can efficiently infect mature, postmitotic neurons. Methods for manipulating the vectors and transfecting CNS cells are well known (see, e.g., Kennedy and Steiner; Yung). A number of studies describe methods for transplanting genetically modified cells into different regions of the brain (Malim, et al.; Rossi and Sarver; Sullenger, et al.; Morgan, et al.; Chatterjee, et al.; Malin, et al.; Hope, et al.). Studies utilizing direct injection of vectors into CNS tissue have also been performed (e.g., Zhang, et al.).
Naked DNA Iniection
Plasmids bearing chimeric genes of the present invention may be purified and injected directly into a target tissue, as exemplified in Example 2 for rat cardiac tissue. The data discussed in Example 2 demonstrate that cardiac injection of plasmid suspended in saline buffer is effective to result in expression of the plasmid in the cardiac cells. Similar approaches have been used successfully by others to express, for example, exogenous genes in rodent cardiac and skeletal muscle (Wolf, et al.; Ascadi, et al., 1991a; Ascadi, et al., 1991b; Lin, et al.; Kitsis, et al..
Liposome-Mediated Gene Transfer
Liposomes may be employed to deliver genes to target tissues using methods known in the art. The liposomes may be constructed to contain a targeting moiety or ligand, such as an antigen, an antibody, or a virus on their surface to facilitate delivery to the appropriate tissue. For example, liposomes prepared with ultraviolet (UV) inactivated Hemagglutinating Virus of Japan (HVJ) may be used to deliver DNA to selected tissues (Morishita, et al.).
The liposomes may also be surface-coated, e.g., by incorporation of phospholipid--polyethyleneglycol conjugates, to extend blood circulation time and allow for greater targeting via the bloodstream. Liposomes of this type are well known.
Receptor-Mediated Gene Transfer
Receptor-mediated endocytic pathways for the uptake of DNA may permit the targeted delivery of genes to specific cell types in vivo. Receptor-mediated methods of gene transfer involve the generation of complexes between plasmid DNA and specific polypeptide ligands (Wu) that can be recognized by receptors on the cell surface. One of the problems with receptor-mediated uptake for gene delivery is that the endocytic vesicles formed during this process may be transported to the lysosome, where the contents of the endosome are degraded. Methods have been developed to facilitate escape of the DNA from the endosome during the course of its transport. For example, either whole adenovirus (Wagner, et al., 1992a; Christiano, et al.) or fusogenic peptides of the influenza HA gene product (Wagner, et al., 1992b) may be used to induce efficient disruption of DNA-containing endosomes.
Administration of Constructs
In cases such as those outlined above, where a vector may be targeted to selectively transfect a specific population of cells, it will be understood that in addition to local administration (such as may be achieved by injection into the target tissue), the vector may be administered systemically (e.g., intravenously) in a biologically-compatible solution or pharmaceutically acceptable delivery vehicle. Vector constructs administered in this way may selectively infect the target tissue. According to the present invention, the presence of a target tissue-specific promoter on the construct provides an independent means of restricting expression of the therapeutic gene.
VIII. Applications
A. Therapeutic Applications
Compositions and methods of the present invention may be useful to prevent tissue damage and/or death, due to ischemia and/or subsequent reperfusion, in a variety of tissues. As stated above, an exemplary application is in the reduction of damage due to recurrent myocardial ischemia following a heart attack. The expression of therapeutic genes in the cardiac tissue of heart attack victims may decrease the risk of injury to the tissue during any subsequent ischemic episodes.
Similarly, subjects who have been diagnosed with transient cerebral ischemia, blood clots or other risk factors for stroke may benefit from the use of hypoxia-inducible brain-specific constructs. Subjects diagnosed with acute or chronic renal failure are at greater risk for further ischemic damage to the kidneys (e.g., Rosenberg and Paller). Such subjects may benefit from a therapeutic gene under the control of a kidney-specific promoter, expression of which is enhanced by hypoxic conditions. A variety of other tissues diagnosed as "at risk" for ischemia may be similarly protected, as will be appreciated by one of skill in the art having the benefit of the present specification.
In addition to the utilities discussed above, compositions (e.g., expression vectors containing chimeric genes of the present invention) and methods of the present invention also have a number of applications in animal medicine. Although animals do not usually develop classical atherosclerosis, cardiomyopathies are very common. A number of species develop ischemia-related syndromes, including arteritis, vasculitis, and related vasculopathies, that result in direct redox damage to cells and tissues, particularly to vascular walls and myocardial tissues. Such conditions may be alleviated by administration of chimeric genes of the present invention.
A common and serious condition in horses and ponies involves ascending colonic ischemia, usually caused by strangulation obstruction (Dabareiner, et al.; Sullivan, et al.; Wilson and Stick). A related disease in dogs is called gastric dilation-volvulus (Lantz, et al.). Treatment of these disorders typically involves surgical removal of the obstruction. Reperfusion following such surgery can result in significant injury to reperfused tissues, and typically triggers an inflammatory response with progressive tissue necrosis. The reperfusion may also results in death of the animal due to cardiogenic shock. Compositions and methods of the present invention may be used therapeutically to treat such conditions, and to provide protection to vulnerable tissues, including heart and vascular endothelium, during the treatment of the above syndromes.
Another utility of the present invention is the treatment of cardiac disease in cats and dogs (Miller, et al.). A variety of forms of cardiovascular disease have been described in both cats and dogs, including dilated cardiomyopathy, left ventricular hypertrophy, and hyperthyroidism (Fox, et al.; Atkins, et al.). Systemic necrotizing vasculitis, a condition that may be analogous to atherosclerosis in humans (with regard to plaque formation and intimal proliferation), has been described in Beagles (Scott-Moncrieff, et al.). Each of these conditions may involve ischemia and reperfusion redox injuries to cardiac and vascular tissue that may be treated using the methods and compositions of the present invention.
B. Reporter Constructs for Diagnostic Applications
The present invention may also be employed in diagnostic applications, where it is desirable to localize the site of hypoxia or anoxia. According to this aspect of the invention, therapeutic genes are replaced by reporter genes, such as those used in experiments performed in support of the present invention (e.g., luciferase). The chimeric genes containing the reporter genes under the control of a selected promoter and a hypoxia response element are introduced into a tissue where it is desirable to localize the site of hypoxia. Hypoxia is localized by increased expression of the reporter gene.
The following examples illustrate but in no way are intended to limit the present invention.
MATERIALS AND METHODS
Unless indicated otherwise, chemicals and reagents were obtained from Sigma Chemical Company (St. Louis, Mo.) or Mallinckrodt Specialty Chemicals (Chesterfield, Mo.), restriction endonucleases were obtained from New England Biolabs (Beverly, Mass.), and other modifying enzymes and biochemicals were obtained from Pharmacia Biotech (Piscataway, N.J.), Boehringer Mannheim (Indianapolis, Ind.) or Promega Corporation (Madison, Wis.). Materials for media for cell culture were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO (Detroit, Mich.). Unless otherwise indicated, manipulations of cells, bacteria and nucleic acids were performed using standard methods and protocols (e.g., Titus; Sambrook, et al.; Ausubel, et al.).
A. Definitions
"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Several transformation methods are commonly used in the art, and may be found, for example, in Ausubel, et al., and Sambrook, et al.
"Transfection" refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO4 and electroporation. Successful transfection is generally recognized when any indication of the operation of the expression vector occurs within the host cell.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences (restriction sites) in the DNA. The various restriction enzymes used herein are commercially available (e.g., New England Biolabs, Beverly, Mass.) and their reaction conditions are known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of a plasmid or of a DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 10 μg of DNA are digested with about 20 to 40 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about one hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion, the reaction products are run on a gel (e.g., agarose) to isolate desired fragments.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (e.g., Sambrook, et al.). Unless otherwise noted, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.
"Filling" or "blunting" refer to the procedures by which the single stranded end in the cohesive terminus of a restriction enzyme-cleaved nucleic acid is converted to a double strand. This eliminates the cohesive terminus and forms a blunt end. This process is a versatile tool for converting a restriction cut end that may be cohesive with the ends created by only one or a few other restriction enzymes into a terminus compatible with any blunt-cutting restriction endonuclease or other filled cohesive terminus. Typically, blunting is accomplished by incubating 2-15 μg of the target DNA in a buffer containing 10 mM MgCl2, 1 Mm dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) at about 37° C. in the presence of 8 units of the Klenow fragment of DNA polymerase I (Boehringer Mannheim, Indianapolis, Ind.) and 250 μM of each of the four deoxynucleoside triphosphates (Boehringer Mannheim). The incubation is generally terminated after about 30 min. The reaction products may be purified using standard phenol and chloroform extraction methods followed by ethanol precipitation.
"Northern" blotting is a method by which the presence of a cellular MRNA is confirmed by hybridization to a known, labelled oligonucleotide, DNA or RNA fragment. For the purposes herein, unless otherwise provided, Northern analysis shall mean electrophoretic separation of RNA, typically MRNA, on agarose (e.g., 1%) in the presence of a denaturant (e.g., 7% formaldehyde), transfer to nitrocellulose or nylon membrane, hybridization to the labelled fragment, washing, and detection of the labeled fragment, as described by Sambrook, et al.
B. Cells and Media
HeLa cells, Hep G2 cells and C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC; Rockville, Md.). Human arterial endothelial cells were obtained from Clonetics Corp. (San Diego, Calif.). Unless otherwise indicated, the cells were grown at 37° C. under 5 or 10% CO2 in MEM or DMEM medium (Gibco/BRL) containing 10% fetal bovine serum (Gibco/BRL).
Cardiac myocytes were isolated and cultured as described previously (Bishopric, et al., Webster and Bisphopric, 1992). Briefly, hearts from about 30 (three litters) were minced and subjected to serial trypsin digestion to release single cells. After the final digestion, the cells were washed and preplated for 0.5 h in minimal essential medium (MEM; Gibco/BRL, Gaithersburg, Md.) with 5% fetal calf serum (FCS; Gibco/BRL). Nonattached cells were re-plated in 60-mm Falcon dishes (Becton Dickinson Labware, Lincoln Park, N.J.) at a density of about 2.5×106 cells per dish in MEM containing 5% fetal calf serum, 2.0 g/l glucose and 10 mM HEPES, and grown at 37° C. under 5 or 10% CO2.
C. DNA
1. Therapeutic Genes
Bcl-2 cDNA was obtained in the expression vector pSFFV-Bcl-2 from Dr. Stanley Korsemeyer (Washington University, St. Louis, Mo.; Hockenbery, et al., 1990). Nitric oxide synthase (bNOS) cDNA was obtained from Dr. Solomon Snyder in the vector pNOS (Johns Hopkins University, Baltimore, Md.; Bredt, et al., 1991).
2. Promoters
(i) Cardiac-specific
pαMHC5.5 CAT, containing 5.5 kilobases (Kb) 5' of the mouse α-myosin heavy chain (αMHC) promoter ligated to the chloramphenicol acetyl transferase (CAT) gene, was obtained from Dr. Jeffrey Robbins (University of Cincinnati, College of Medicine, Cincinnati, Ohio; Subramaniam, et al.).
pαMHC2.0 CAT, containing 2.0 Kb of the rat αMHC promoter ligated to the CAT gene, was obtained from Dr. Thomas Gustafson (University of Maryland, Baltimore, Md.; Gustafson, et al.).
pαMHC86 CAT, containing 86 base pairs (bp) of the rat αMHC promoter ligated to the CAT gene, was obtained from Dr. Bruce Markham (Medical College of Wisconsin, Milwaukee, Wis.). The construct was made by 5' truncation of pαMHC2.0CAT and blunt end ligation to the CAT gene. The sequence of the 86 bp promoter fragment is provided herein as SEQ ID NO:24.
pHCA118 CAT, containing 118 bp of the region 5' of the human cardiac α-actin promoter ligated to the CAT gene, was also obtained from Dr. Larry Kedes (Minty and Kedes).
(i) Skeletal muscle-specific
pHSA-150CAT, containing 150 bp of the human skeletal muscle α-actin promoter ligated to the CAT gene, was obtained from Dr. Larry Kedes (University of Southern California, Los Angeles, Calif.; Muscat and Kedes).
3. Hypoxia Response Elements
A construct containing four tandem copies of the erythropoietin gene 3' hypoxia inducible enhancer element cloned into the BamHI site of pGEM-4Z (Promega Corp., Madison, Wis.) was obtained from Dr. Greg Semenza (Johns Hopkins University School of Medicine, Baltimore, Md.; Semenza and Wang, 1992). The enhancer element fragment, termed herein as HREE1 (SEQ ID NO:6), was excised from the pGEM vector by cleavage with SmaI and HincII for blunt end subcloning into constructs of the present invention (below).
A construct containing 691 bp (-628 to +63) of the β-enolase (ENO3) gene was obtained from Dr. Charlotte Peterson (Veterans Administration Medical Center, University of Arkansas, Little Rock, Ark.). A sequence containing this region is presented herein as SEQ ID NO:29.
4. Chimeric Genes and Expression Vectors of the Present Invention
The vector pGL2PV (plasmid-gene-1 lght-promoter-vector; Promega Corp., Madison, Wis.), was used as the base vector for the construction of most of the plasmids described below. pGL2PV is a eukaryotic expression vector containing the SV40 early promoter upstream of the luciferase gene. The vector multiple cloning (MCS) site is just upstream of the SV40 promoter, and is designed for the insertion of DNA fragments containing enhancer sequences. pGL2BV (Promega Corp.) is similar to pGL2PV, but it does not contain an SV40 early promoter.
(i) HREE1/luc Constructs with Different Tissue-Specific Promoters
Plasmid pGLHRE (FIGS. 1B, 2A, 3A) was made by blunt-1 lighting the 240 bp HREE1 fragment (SEQ ID NO:6) into the SmaI site of the MCS of pGL2PV (FIG. 1A).
Plasmid pGLHSA-150HRE (FIG. 2B) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 150 bp HindIII-SmaI fragment from pHSA-150CAT containing a fragment of the human skeletal actin (HSA) promoter.
Plasmid pGLαMHC86 HRE (FIG. 2C) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 120 bp HindIII-EcoRI fragment from pαMHC86 CAT containing 86 bp (SEQ ID NO:24) of the human α-myosin heavy chain (α-MHC) promoter. The EcoRI end of the 120 bp fragment was filled in with DNA polymerase I using standard methods (Sambrook, et al.) before blunt end ligation to the vector SmaI site.
Plasmid DGLαMHC86 -GATA-HRE was made by cloning a 36 bp oligonucleotide (SEQ ID NO:1; described above), containing a duplicated GATA 4 box into the HindIII site (filled in with polymerase) of plasmid pGLαMHC86 HRE, upstream of the 86 bp promoter fragment.
Plasmid pGLHCA118 HRE (FIG. 2D) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 188 bp HindIII-EcoRI fragment from pHCA118 CAT, containing 118 bp of the human cardiac actin (HCA) promoter plus 70 bp of actin exon 1. The EcoRI end of the 188 bp fragment was filled in with DNA polymerase I as above before blunt end ligation to the vector SmaI site.
Plasmid pGLαMHC1.2 HRE (FIG. 3B) was made by digesting pGLHRE with HindIII and SmaI to drop out the SV40 promoter and replacing it with a 1.2 kb HindIII-EcoRI fragment from pαMHC20 CAT containing 1.2 kb of the human α-MHC promoter. The EcoRI end of the 1.2 kb fragment was filled in as above in prior to cloning.
(ii) PKM Promoter/luc Constructs
Plasmid pGLPKM460, containing 460 bp of the rat muscle specific pyruvate kinase (PKM) gene promoter and 140 bp of the PKM coding sequence (SEQ ID NO:7), was created using polymerase chain reaction (PCR) as follows. PKM-specific primers containing endonuclease restriction sites near their 5' end were designed based on the nucleotide sequence of the PKM gene (Takenaka, et al., 1989). PKM primer F (SEQ ID NO:2) contained a KpnI site, while PKM primer R (SEQ ID NO:3) contained a XhoI site. PCR was carried out using the above primers and 1 μg of rat heart genomic DNA as a template for 25 cycles using standard procedures and a Perkin-Elmer (Norwalk, Conn.) DNA thermal cycler. The PCR product (FIG. 6A) was purified by agarose gel electrophoresis, cut with KpnI and XhoI, and cloned into KpnI/XhoI cut pGL2BV (FIG. 6B; Promega Corp., Madison, Wis.), generating pGLPKM460 (FIG. 6C).
Plasmid pGLPKM285 (FIG. 6E) was generated by digesting pGLPKM460 with SmaI to drop out the -460 to -285 portion of the promoter, and religating the vector. pGLPKMD (FIG. 6D) was generated by digesting pGLPKM460 with SmaI to isolate the -460 to -285 portion of the promoter, and cloning that fragment into pGL2PV (Promega Corp.) that had been cut with SmaI.
(iii) Et-1 Promoter/luc Constructs
Plasmid pGLET-1700 (FIG. 7C), containing 700 bp of the human ET-1 gene promoter (SEQ ID NO:8), was created using PCR to amplify HeLa cell genomic DNA as described above. ET-1 specific primers were designed based on the promoter sequence (Inoue, et al., 1989) of the ET-1 gene. The forward primer (SEQ ID NO:4) contained PstI and KpnI sites, while the reverse primer (SEQ ID NO:5) contained HindIII and XbaI sites. The PCR product (FIG. 7A) was purified by gel electrophoresis, cut with KpnI and HindIII, and cloned into KpnI/HindIII cut pGL2BV (FIG. 7B; Promega Corp.).
(iv) ENO3 Promoter/luc Constructs
Plasmid pGLENO628 was constructed by cloning a blunt ended genomic DNA containing an ENO3 promoter fragment (-628 to +63; SEQ ID NO:29), isolated from a lambda gt10 human genomic library, into the SmaI site of pGL2BV.
(v) Therapeutic Gene Constructs
Plasmid pαMHC1.2 HRE-NOS (FIG. 4B) was made by digesting plasmid pGLαMHC1.2 HRE (FIG. 4A) with HindIII and EcoRV to drop out the luciferase cDNA and replacing it with a HindIII/XbaI fragment from pNOS containing a full length NOS CDNA.
Plasmid pαMHC1.2 HRE-Bcl-2 (FIG. 5B) was made by digesting pSFFV-Bcl-2 with SalI, blunting the vector as described above, removing the SFFV promoter from the linearized vector with an EcoRI digest, and replacing the SFFV promoter with a SmaI/EcoRI fragment from pgLαMHC1.2 HREE containing the 1.2 kb αMHC promoter fragment and the 240 bp HREE1.
(vi) Other Plasmid Constructs
Plasmid pαMHC5.5 HRE-CAT was made by inserting the 240 bp HREE1 immediately 5' of the AMHC promoter of pαMHC5.5 CAT.
(vi) Adenoviral Constructs
Adenoviral constructs are made using standard methods (e.g., Friedman,, et al., 1986; Hertz and Gerard, 1993), as follows.
Construct AdαMHC1.2Bcl2HREE is made by inserting a 3.34 Kb EcoRI/HindIII fragment from pαMHC1.2-Bcl-2 (containing 1.2 Kb of the α-MHC promoter, 1.9 Kb Bcl-2 CDNA, and 240 bp HREE1) into pAPLCMV digested with EcoRI and HindIII to drop out the CMV promoter and CAT gene. pAPLCMV, which may be obtained from Dr. Larry Kedes (University of Southern California, Los Angeles, Calif.; Kass-Eisler, et al., 1993), is a base replication deficient adenoviral expression vector. The backbone adenoviral vector for recombination, p9M17, may also be obtained from Dr. Larry Kedes.
Recombinant pAPLCMV (pAdαMHC1.2bcl-2HRE) and p9M17 are used to co-transfect 293 cells (ATCC) to propagate the adenovirus.
EXAMPLE 1 Tissue Specific Hypoxia Induced Expression In Vitro
Constructs pGLHRE, pGLHSA-150HRE, pαMHC5.5 HRE-CAT, pGLαMHC1.2 HRE, pGLHCA118 HRE and pGL-Eno628 were tested for tissue-specific expression and hypoxia inducibility in HeLa cells, Hep G2 cells, differentiated C2C12 muscle myotubes, and cardiac myocytes.
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A.      Buffers and Solutions                                             
        HEPES buffered saline (HeBS; 2X solution)                         
16.4 g         NaCl                                                       
11.9 g         HEPES acid                                                 
0.21 g         Na.sub.2 HPO.sub.4                                         
H.sub.2 O to 1 liter                                                      
Titrate Ph to 7.05 with 5 M NaOH.                                         
PBS Buffer                                                                
137 mM         NaCl                                                       
2.7 mM         KCl                                                        
4.3 mM         Na.sub.2 HPO.sub.4                                         
1.4 mM         KH.sub.2 PO.sub.4                                          
Adjust pH to 7.1.                                                         
Reconstituted Luciferase Assay Reagent (LAR)                              
20 mM          Tricine                                                    
1.07 mM        (MgCO.sub.3).sub.4 MG(OH).sub.2.5H.sub.2 O                 
2.67 mM        MgSO.sub.4                                                 
0.1 mM         EDTA                                                       
33.3 mM        DTT                                                        
270 μM      coenzyme A                                                 
470 μM      luciferin                                                  
530 μM      ATP                                                        
Cell Culture Lysis Reagent (CCLR; 1X Solution)                            
25 mM          Tris-phosphate, pH 7.8                                     
2 mM           DTT                                                        
2 mM           1,2-diaminocyclohexane-N,N,N',N'-                          
               tetraacetic acid                                           
10%            glycerol                                                   
1%             Triton X-100                                               
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A. Cell Transfection
HeLa cells, C2C12 myocytes, and cardiac myocytes were transfected with the indicated plasmid DNA by the standard calcium phosphate procedure (Ausubel, et al.).
Briefly, 105 cells were plated on a 10-cm tissue culture dish and grown for 3 days. The cells were split 1:10 into 10 ml of medium one day before application of plasmid DNA. DNA for transfection was prepared by resuspending an ethanol-precipitated pellet containing 20 μg of the plasmid DNA in 450 μl ddH2 O and adding 50 μl of 2.5 mM CaCd2.
500 μl of 2× HeBS were added to a 15 ml conical centrifuge tube, and the solution was aerated by bubbling air with a 10 ml pipette attached to an automatic pipettor (Drummond Instruments, Fisher Scientific, Pittsburgh, Pa.). The DNA/CaCl2 solution was added dropwise, and the resultant mixture was vortexed for 5 seconds and then allowed to sit for 20 minutes at room temperature to form precipitate.
The precipitate was added to the dishes containing the cells and the dishes were incubated overnight.
The cells were washed twice with 5 ml PBS and fed with 10 ml of complete medium. The cells were then allowed to recover for 24 hours before incubation under an atmosphere of 1.0% O2, 5% CO2, 94% N2 for an additional 20 hours.
B. Exposure to Hypoxic Conditions
Two to three days after transfection, the cells were exposed to atmospheric oxygen (approximately 21% O2, 5% CO2, balance N2 ; pO2 =˜160 mmHg), or to hypoxic conditions (approximately 0.5-2.0% O2, 5% CO2, balance N2 ; pO2 =˜4-8 mmHg) in an environmental chamber (Anaerobic Systems, San Jose, Calif., USA) which was equipped with a Nikon TMS microscope and a continuous readout oxygen electrode (Controls Katharobic, Philadelphia, Pa., USA). Unless otherwise indicated, the cells were kept in the chambers for one day prior to assaying for luciferase expression.
C. Luciferase Expression
Cells transfected and treated as above were assayed for expression of the luciferase enzyme using a standard reaction protocol (Titus). Briefly, 1 ml of CCLR and 1 ml of LAR were allowed to equilibrate at room temperature. The culture medium in the dish containing the cells to be assayed was removed and the cells were rinsed twice in PBS buffer.
Approximately 300 μl of the room-temperature CCLR was added to the dish containing the cells, and the dish was incubated at room temperature for 10-15 minutes. The cells were then scraped off the bottom of the culture dish, and the solution containing the cells was transferred to a micro-centrifuge tube. The tube was centrifuged in a table-top microcentrifuge briefly (about 5 seconds) to pellet large debris.
20 μl of the supernatant (cell extract) were mixed with 100 μl of LAR at room temperature, and the light produced was measured for a period of 5 minutes, starting approximately 5 seconds after mixing, with a model # 1250 LKB luminometer (Bioorbit, Gaithersburg, Md.).
D. Results
Data from HeLa, C2C12, and cardiac cells are given in Table 1, below. Values, presented in arbitrary units, represent averages of three or more experiments for each condition.
                                  TABLE 1                                 
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REGULATED EXPRESSION OF UBIQUITOUS-MUSCLE-                                
AND CARDIAC-SPECIFIC PROMOTERS BY HYPOXIA                                 
GL2PV     GLHRE  GLHSA.sub.150 HRE                                        
                         αMHC.sub.1.2 HRE                           
                                GLHCA.sub.118 HRE                         
                                        GLENO.sub.628                     
A      Hx A  Hx  A   Hx  A  Hx  A   Hx  A  Hx                             
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HeLa                                                                      
    18 27  56                                                             
             387 BG      BG     BG      --                                
C2Cl2                                                                     
    189                                                                   
       204                                                                
          350                                                             
             1680                                                         
                 46  278 BG     48  248 320                               
                                           1560                           
Cardiac                                                                   
    24 27 22 165 18  94  21 85  38  263 210                               
                                           1610                           
__________________________________________________________________________
 BG  Background
Data shown in the table demonstrate that (i) none of the tested constructs carrying tissue-specific promoters are expressed above background in fibroblast-derived HeLa cells under either normal or hypoxic conditions, (ii) cells containing constructs having HREE1 and a compatible promoter (including the SV40 and tissue-specific promoters) express the reporter at levels that are ˜5 to ˜7 times greater under hypoxic conditions than under aerobic conditions; (iii) the HREE1 element is equally active in different cells and independent of the promoter; (iv) the α-MHC1.2 promoter expresses in cardiac, but not in skeletal or fibroblast-derived cells, the HCA118 promoter expresses in both cardiac and skeletal muscle cells, but not in fibroblast-derived cells, and the HSA150 promoter expresses in both skeletal and cardiac muscle, with stronger expression in skeletal muscle; and (v) basal (aerobic) expression from α-MHC1.2 HCA118, and HSA150 promoters is weak.
These results indicate that the HREE1 element is fully functional when fused to muscle and cardiac specific promoters and that muscle and cardiac cells are fully responsive to hypoxia in terms of the regulation of these promoters, and suggest that the αMHC 1 2 promoter is an exemplary promoter for moderate levels of cardiac-specific expression.
The data also show that both the HREE present in the ENO3 promoter and HREE1, when present in constructs with the SV40 promoter, result in comparable levels of hypoxia induction in skeletal muscle cells. In cardiac cells, however, constructs containing the ENO3 HREE are expressed at significantly higher levels than those containing HREE1. Further, hypoxia increases the level of expression of the ENO3 HREE containing constructs in cardiac cells by over seven-fold, as compared with less than 5-fold in skeletal muscle cells. Plasmid pGLENO628 confers induced expression in C2C12 myotubes and cardiac myocytes that is at least equivalent to four copies of the erythropoietin HRE (HREE1) in these cells. These results suggest that the HREE in the ENO3 promoter fragment may be a particularly effective HREE for hypoxia induction in constructs targeted with a tissue-specific promoter to cardiac or skeletal muscle cells.
EXAMPLE 2 Tissue Specific Hypoxia Induced Expression In Vivo Following Injection of Constructs into Target Animal Tissue
Constructs of the present invention were injected directly into cardiac tissue using techniques described in Buttrick, et al., (1992) and Buttrick, et al., (1993). Briefly, adult female Wistar rats were anesthetized with an intraperitoneal injection of chloral hydrate (0.7 ml/100 g of a 4% solution). Cardiac injections were made directly into the apex of the heart through a lateral thoractomy, after which the heart was replaced in the chest, the rats were briefly hyperventilated, and the incision closed. Fifty microliters of a DNA solution containing 2 μg/μl of either pGLHRE or pGLHCA118 HRE in 20% sucrose and 2% Evans blue were injected through a 27-gauge needle. Following injection the rats were subjected to a 20 min ischemia by cannulation of the coronary artery as described by Smith, et al. (1988).
Hypoxia-inducibility of vector expression was assayed as follows. Hearts were excised approximately 20 hours after the induced ischemia and the ventricles were washed with ice-cold phosphate buffered saline (PBS). The tissue was suspended in 1 ml of ice-cold PBS containing 20% sucrose and homogenized with a Polytron (Kinematica, Switzerland) for 45 sec. After centrifugation at 10,000×g for 10 min supernatants were analyzed for luciferase expression by the assay method described above. Protein was measured using a BioRad assay kit (BioRad Laboratories, Hercules, Calif.).
The results of the experiments are shown in Table 2, below. Luciferase expression in hearts from rats injected with pGLHRE or pGLHCA118HRE and subjected to ischemia was approximately 2-fold higher than expression in hearts from control animals injected with saline (n=3).
              TABLE 2                                                     
______________________________________                                    
ISCHEMIA INDUCIBLE EXPRESSION OF pGLHRE AND                               
pGLHCA.sub.118 HRE IN RAT HEART                                           
             Luciferase Activity Light                                    
             Units/mg Protein                                             
Plasmid        Aerobic 20 min. Ischemic                                   
______________________________________                                    
pGLHRE         1180    2440                                               
pGLHCA.sub.118 HRE                                                        
               88       127                                               
Control        15       21                                                
______________________________________                                    
 Rat hearts were injected with plasmids as described above. A 20 min.     
 ischemia was imposed on one group (3 rats) and the other (1 control) was 
 sham operated. Tissue samples were harvested and assayed for luciferase  
 expression 20 hr. later.
These results indicate that the direct injection of plasmid DNA, made in accordance with the teachings of the present specification, into hearts of living mammals is effective to result in the expression of genes carried on those plasmids. Further, these results indicate that expression vectors carrying chimeric genes of the present invention are effective to result in significantly increased levels of expression in response to hypoxia caused by ischemia in vivo.
EXAMPLE 3 Stable Expression of Hypoxia Regulated NOS and Bcl-2 Genes In Vitro
106 C2C12 myoblasts were cotransfected with pSV2Neo (Minty and Kedes) and a test plasmid at a ratio of 1:19 (1 μg pSV2Neo+19 μg test plasmid) using standard methods (Minty and Kedes, 1986). Test plasmids were pGLHRE, pGLHCA118 HRE, pGLαMHC1.2 HRE, pSFFV-Bcl-2, and pNOS-HRE. Cultures were selected on day 2 following transfection with 400 μg/ml of the neomycin drug G418 (Gibco/BRL). Colonies of cells resistant to G418 appeared after 10 to 14 days. The resistant cells were pooled. Mass cultures were assayed for the expression of luciferase as described above or by Northern blot assay (Webster, et al., 1993) for the expression of Bcl-2 or NOS RNA. Stable lines were positive for expression of the transfected genes.
Mass cultures were subjected to differentiation conditions by transferring them to low mitogen medium (DMEM with 2% horse serum) and were analyzed visually for differentiation into myotubes. There was no apparent difference between transfected and control cells. Approximately 40% of cells were fused into multinucleate myotubes after 24 h in low mitogen medium. All cultures contained approximately 74% myotubes after 48 h.
Reporter-transfected cells differentiated normally and respond to hypoxia with the predicted induction of reporter. NOS-transfected cells appeared normal both during the hypoxia and during subsequent reoxygenation. A stable line of C2C12 cells that constitutively over-expresses Bcl-2 (without HREE1) was also constructed as described above, and the cells showed normal growth and differentiation characteristics.
Taken together, the data presented above suggest that inclusion of HRE elements, Bcl-2 over-expression, and hypoxia-induced over-expression of NOS is not toxic to muscle cells in vitro. Further, the data indicate that the cells may be protected from the deleterious effects of hypoxia by the expression of therapeutic genes (e.g., NOS).
EXAMPLE 4 Expression of DGLPKM Plasmids under Hypoxic Conditions
Plasmid pGLPKM460 was transfected into C2C12 cells and cardiac myocytes and assayed for luciferase activity as described in Example 1. The expression of PGLPKM in both transfected C2C12 myotubes and neonatal cardiac myocytes was increased by 6±2 fold (n=4) in both cell types by incubation of the cells in an atmosphere containing 0.5% O2, 5% CO2, balance N2 (hypoxic conditions) relative to normal conditions, as described in Example 1.
A portion of this HRE element, obtained by digesting with SmaI to cut at an internal SmaI site, is also effective as a hypoxia response enhancer element. This fragment, termed HREPKM285, confers hypoxia-induced expression in C2C12 myotubes and cardiac myocytes similar to that obtained with pGLPKM460. This level of hypoxia induction is at least equivalent to that obtained using HREE1 (SEQ ID NO:6).
These results indicate that the PKM promoter fragment contained in the sequence represented as SEQ ID NO:7 contains an HRE element that is effective at enhancing the expression of chimeric genes containing the element under conditions of hypoxia.
The PKM promoter sequence has no significant homology with the erythropoietin HRE consensus, but does share a consensus sequence (SEQ ID NO:31) with the ENO3 promoter fragment (SEQ ID NO:29). This consensus, located approximately 88 bp upstream of the transcription start site of PKM and approximately 70 bp upstream of the transcription start site of ENO3, may represent an important element for conferring enhancement of expression in response to hypoxia.
EXAMPLE 5 Expression of pGLET-1700 Plasmids under Hypoxic Conditions
Plasmid pGLET-1700 was transfected into human arterial endothelial cells as described in Example 1. The expression of pGLET-1700 in these cells was increased 5 fold by incubation of the cells in a hypoxic atmosphere as described above. No significant induction of pGLET-1700 was observed in any other cell types tested, including HeLa, C2C12, and cardiac myocytes. Elements contained within the 700 bp sequence have no significant homology with the erythropoietin HRE consensus.
These results indicate that the 700 bp fragment of the human ET-1 gene promoter corresponding to the sequence represented herein as SEQ ID NO:8 is effective to (i) restrict expression of genes under its control to the vascular endothelium, and (ii) confer hypoxia-inducibility on the expression of those genes. Accordingly, this fragment, in conjunction with a therapeutic or reporter gene, may be used in the methods of the present invention to both target expression to a selected tissue (vascular endothelium), and confer enhancement of expression by hypoxia.
While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.
__________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 31                                             
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 35 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: GATA4 Enhancer                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
CAAAGGGCCGATGGGCAGATAGAGGAGAGACAGGA35                                     
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 33 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: PKM primer F                                      
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
AATTGGTACCCGGGCGAGCGCCGGGAGGGTGGA33                                       
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 32 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: PKM primer R                                      
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
TTAACTCGAGGCACTATGGCATTGGCTCTGGG32                                        
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 41 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: ET-1 primer F                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
TATATCTGCAGGTACCGATAGGGAAAAGACTGGCATGTGCC41                               
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 43 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: ET-1 primer R                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
TATATAAGCTTCTAGAGACCCGTTCGCCTGGCGCGCAGATGCA43                             
(2) INFORMATION FOR SEQ ID NO:6:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 240 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: HREE1 (Hypoxia responsive enhancer                
element 1)                                                                
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
CGGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCCGGGATC60            
CCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCCGGGATC120           
CCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCCGGGATC180           
CCGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCCGATCCCG240           
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 560 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: sequence containing PKM promoter frag.            
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
GAGTCACCGGGCGGGGCTGGAGGAATGTCCGGGACCTATAAATCTGGGCAACGCCCTGGT60            
AGGCCAGGGCAGATGGGGCACCTGGGCAGAATTCCAAAATGGGATTATGTAGCCTCTGAG120           
GTCCTAAAGCAACAGGTGGCGGACCACCCGGGGATCTAGGGGTGGTGGCGGCGGTGGACC180           
CGAGGGCGGGTCCTGCCTCCTCACCACTTCCCCATTGGCCATCAGAATGACCCATGCGCA240           
ATTTTGGTTTGCAATGTCCTTCCGCCACGGAAGGTAGTCCCCCTCAAAAGGGCAACCTGC300           
TTGTCCCGCCTACCCTGCGACTCTCTCAGAAGGTGCGGGTGCCTGTTGAGAGGCGGGGCT360           
CTGCTAGCTCCTGCCCGGATTGGGCGAGGGGCGGGGCTGCGGAGGGATTGCGGCGGCCCG420           
CAGCAGTGATAACCTTGAGGCCCAGTCTGCGCAGCCCCGCACAGCAGCGACCCGTCCTAA480           
GTCGACAGACGTCCTCTTTAGGTATTGCAACAGGATCTGAAGTACGCCCGAGGTGAGCGG540           
GGAGAACCTTTGCCATTCTC560                                                   
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 713 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: Sequence containing ET-1 promoter                 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                   
GATAGGGAAAAGACTGGCATGTGCCTAAACGAGCTCTGATGTTATTTTTAAGCTCCCTTT60            
CTTGCCAATCCCTCACGGATCTTTCTCCGATAGATGCAAAGAACTTCAGCAAAAAAGACC120           
CGCAGGAAGGGGCTTGAAGAGAAAAGTACGTTGATCTGCCAAAATAGTCTGACCCCCAGT180           
AGTGGGCAGTGACGAGGGAGAGCATTCCCTTGTTTGACTGAGACTAGAATCGGAGAGACA240           
TAAAAGGAAAATGAAGCGAGCAACAATTAAAAAAAATTCCCCGCACACAACAATACAATC300           
TATTTAAACTGTGGCTCATACTTTTCATACCAATGGTATGACTTTTTTTCTGGAGTCCCC360           
TCTTCTGATTCTTGAACTCCGGGGCTGGCAGCTTGCAAAGGGGAAGCGGACTCCAGCACT420           
GCACGGGCAGGTTTAGCAAAGGTCTCTAATGGGTATTTTCTTTTTCTTAGCCCTGCCCCC480           
GAATTGTCAGACGGCGGGCGTCTGCTTCTGAAGTTAGCAGTGATTTCCTTTCGGGCCTGG540           
CTTATCTCCGGCTGCACGTTGCCTGTTGGTGACTAATAACACAATAACATTGTCTGGGGC600           
TGGAATAAAGTCGGAGCTGTTTACCCCCACTCTAATAGGGGTTCAATATAAAAAGCCGGC660           
AGAGAGCTGTCCAAGTCAGACGCGCCTCTGCATCTGCGCCAGGCGAACGGGTC713                  
(2) INFORMATION FOR SEQ ID NO:9:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 118 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: HCA118 promoter fragment                          
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                   
CGAAGGGGACCAAATAAGGCAAGGTGGCAGACCGGGCCCCCCACCCCTGCCCCCGGCTGC60            
TCCAACTGACCCTGTCCATCAGCGTTCTATAAAGCGGCCCTCCTGGAGCCAGCCACCC118             
(2) INFORMATION FOR SEQ ID NO:10:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1588 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: Rat alpha MHC promoter fragment                   
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                  
GAATTCTCTTACTATCAAAGGGAAACTGAGTCATGCACCTGCAAAATGAATGCCCTCCCT60            
GGACATCATGACTTTGTCCCTGGGGAGCCAGCACTGTGGAACTCCAGGTCTGAGAGTAGG120           
AGGCACCCCTCAGCCTGAAGCTGTGCAGATAGCTAGGGTGTAAAAGAGGGAAGGGGGGAG180           
GCTGGAATGGGAGCTTGTGTGTTCGGAGACAGGGGACAAATATTAGGCCCGTAAGAGAAG240           
GTGACCCTTACCCAGTGTGTTCAACTCAGCCTTTCAGATTAAAAATAACTAAGGTAAGGG300           
CCATGTGGGTAGGGGAGGTGGTGTGAGACGGTCCTGTCTCTCCTCTATCTGCCCATCGGC360           
CCTTTGGGGAGGAGGAAATGTGCCCAAGGACTAAAAAAGGCCTGGAGCCAGAGGGGCTAG420           
GGCTAAGCAGACCTTTCATGGGCAAACCTCAGGGCTGCTGTCCTCCTGTCACCTCCAGAG480           
CCAAGGGATCAAAGGAGGAGGAGCCAGACAGGAGGGATGGGAGGGAGGGTCCCAGCAGAT540           
GACTCCAAATTTAGGCAGCAGGCACGCGGAATGAGCTATAAAGGGGCTGGAGCGCTGAGA600           
GCTGTCAGACCGAGATTTCTCCATCCCAAGTAAGAAGGAGTTTAGCGTGGGGGCTCTCCA660           
ACCGCACCAGACCTGTCCCACCTAGAGGGAAAGTGTCTTCCCTGGAAGTGGGCTCCTCCC720           
ACCGGCCTGGGAAGATTCCTCGGTGGGCAGGATGTTCTACTGGATGCCCCTTCCCTTCCA780           
CTGCCTCCTCCCTCCCTTGTCTTGATTAATCTTGGCTCTTAGTGTTCAGAAAGATTTGCC840           
CGGTGCTGTCTACTCCATCTGTCTCTACTCTCTCTGCCTTGCCTTCTTGTGTGTTCTCCT900           
TTTCCACGTGTTTCTCACTCCACTGCCTCCCCCCCCCCCTTCATTTTTATCCTTCCTTTC960           
TTTCTGTGTCAGAATGCTGGGAATCAAACCCAGGGCTTCATACACGTCAAGTAAGCAATC1020          
TCCCAGTGAGTCAAAGCTTTAATCCTCTGGGTGCTGTCTTACCGAGCCTCACTCCCTGTC1080          
TTGTCCTGTTCCGTCCTAGTCAGGATCTCTGGTCCGTCTCTCAGCTTCTGCTACTCCTCT1140          
CCCTGCCTGCTCTTCTCTCCGTCCAGCTGCACCTCTGTGGCGCTCATTCCAGCCGTGGTC1200          
CAAATTCTCTGTGAAAAGATTAACCGGGTGAGAATGCCCCCAGTTTCCCCTGTAGACAGC1260          
AGATCATGATTTTCCCCAGAAGCCAGACTTCCAGCGCCCGCCCTCTGCCCAGCAACTTGA1320          
CACTCTTAGCAAACTTCAGCCACCCTTCCCCCACATAGACCAAGTCTTGCAGAGAGCCTT1380          
CCTTCAGATGACTTCGAGTTCTTGCAAAGGAAGGAGAACTCTTTGTGGCGGGGAAGCAGG1440          
CACTTTACACGGAGTCTGACGGGAGGTCATAGGCTATGGCATAGCAGAGGCAGGGAGGTG1500          
GTGGAATTGGACTTCGCGCAGAAGCTAAGCACACACCAGGAATGACATATCCCTCCTATC1560          
TCCCCCATAAGAGTTTAAGAGTGACAGG1588                                          
(2) INFORMATION FOR SEQ ID NO:11:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1679 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: Mouse alpha MHC promoter fragment                 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                  
GAATTCTCTTACTATCAAAGGGAAACTGAGTCGTGCACCTGCAAAGTGGATGCTCTCCCT60            
AGACATCATGACTTTGTCTCTGGGGAGCCAGCACTGTGGAACTTCAGGTCTGAGAGAGTA120           
GGAGGCTCCCCTCAGCCTGAAGCTATGCAGATAGCCAGGGTTGAAAGGGGGAAGGGAGAG180           
CCTGGGATGGGAGCTTGTGTGTTGGAGGCAGGGGACAGATATTAAGCCTGGAAGAGAAGG240           
TGACCCTTACCCAGTTGTTCAACTCACCCTTCAGATTAAAAATAACTGAGGTAAGGGCCT300           
GGGTAGGGGAGGTGGTGTGAGACGCTCCTGTCTCTCCTCTATCTGCCCATCGGCCCTTTG360           
GGGAGGAGGAATGTGCCCAAGGACTAAAAAAAGGCCATGGAGCCAGAGGGGCGAGGGCAA420           
CAGACCTTTCATGGGCAAACCTTGGGGCCCTGCTGTCCTCCTGTCACCTCCAGAGCCAAG480           
GGATCAAAGGAGGAGGAGCCAGGACAGGAGGGAAGTGGGAGGGAGGGTCCCAGCAGAGGA540           
CTCCAAATTTAGGCAGCAGGCATATGGGATGGGATATAAAGGGGCTGGAGCACTGAGAGC600           
TGTCAGAGATTTCTCCAACCCAGGTAAGAGGGAGTTTCGGGTGGGGGCTCTTCACCCACA660           
CCAGACCTCTCCCCACCTAGAAGGAAACTGCCTTTCCTGGAAGTGGGGTTCAGGCCGGTC720           
AGAGATCTGACAGGGTGGCCTTCCACCAGCCTGGGAAGTTCTCAGTGGCAGGAGGTTTCC780           
ACAAGAAACACTGGATGCCCCTTCCCTTACGCTGTCTTCTCCATCTTCCTCCTGGGGATG840           
CTCCTCCCCGTCTTGGTTTATCTTGGCTCTTCGTCTTCAGCAAGATTTGCCCTGTGCTGT900           
CCACTCCATCTTTCTCTACTGTCTCCGTGCCTTGCCTTGCCTTCTTGCGTGTCCTTCCTT960           
TCCACCCATTTCTCACTTCACCTTTTCTCCCCTTCTCATTTGTATTCATCCTTCCTTCCT1020          
TCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTTCTCCCTTCCTTCCTTCCTTCCT1080          
TCCTTCCTTCCTTCCTTCCTTCCTGTGTCAGAGTGCTGAGAATCACACCTGGGGTTCCCA1140          
CCCTTATGTAAACAATCTTCCAGTGAGCCACAGCTTCAGTGCTGCTGGGTGCTCTCTTAC1200          
CTTCCTCACCCCCTGGCTTGTCCTGTTCCATCCTGGTCAGGATCTCTAGATTGGTCTCCC1260          
AGCCTCTGCTACTCCTCTTCCTGCCTGTTCCTCTCTCTGTCCAGCTGCGCCACTGTGGTG1320          
CCTCGTTCCAGCTGTGGTCCACATTCTTCAGGATTCTCTGAAAAGTTAACCAGGTGAGAA1380          
TGTTTCCCCTGTAGACAGCAGATCACGATTCTCCCGGAAGTCAGGCTTCCAGCCCTCTCT1440          
TTCTCTGCCCAGCTGCCCGGCACTCTTAGCAAACCTCAGGCACCCTTACCCCACATAGAC1500          
CTCTGACAGAGAAGCAGGCACTTTACATGGAGTCCTGGTGGGAGAGCCATAGGCTACGGT1560          
GTAAAAGAGGCAGGGAAGTGGTGGTGTAGGAAAGTCAGGACTTCACATAGAAGCCTAGCC1620          
CACACCAGAAATGACAGACAGATCCCTCCTATCTCCCCCATAAGAGTTTGAGTGACAGA1679           
(2) INFORMATION FOR SEQ ID NO:12:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 5057 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: rat bNOS cDNA                                     
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 349..4638                                                   
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                  
ACGTCTGACAAGCTGGTGACCAAGATGCCCAGAGACTAGACCCTATGCTTGTGAGTCACA60            
GTCATCAGACACGGCAAACCTCCAGTCTTCCTGACCTGTTGCTTAGGGACACATCCCGTT120           
GCTGCCCCTGACGTCTGCCTGGTCAACCTTGACTTCCTTTGAGAGTAAGGAAGGGGGCGG180           
GGACACGTTGAAATCATGCCACCCAAGGCCGAATCGGAATGAGCAGATGACGCCAAGTTG240           
ACGTCAAAGACAGAGGCGACAGAAACTCTGCAGCCAGCTCTTGCCCCCGAGGAGCTCAGG300           
TTCCTGCAGGAGTCATTTTAGCTTAGTCTTCTGAAGGACACAGATACCATGGAAGAG357              
MetGluGlu                                                                 
AACACGTTTGGGGTTCAGCAGATCCAACCCAATGTAATTTCTGTTCGT405                       
AsnThrPheGlyValGlnGlnIleGlnProAsnValIleSerValArg                          
51015                                                                     
CTCTTCAAACGCAAAGTGGGAGGTCTGGGCTTCCTGGTGAAGGAACGG453                       
LeuPheLysArgLysValGlyGlyLeuGlyPheLeuValLysGluArg                          
20253035                                                                  
GTCAGCAAGCCTCCCGTGATCATCTCAGACCTGATTCGAGGAGGTGCT501                       
ValSerLysProProValIleIleSerAspLeuIleArgGlyGlyAla                          
404550                                                                    
GCGGAGCAGAGCGGCCTTATCCAAGCTGGAGACATCATTCTCGCAGTC549                       
AlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIleLeuAlaVal                          
556065                                                                    
AACGATCGGCCCTTGGTAGACCTCAGCTATGACAGTGCCCTGGAGGTT597                       
AsnAspArgProLeuValAspLeuSerTyrAspSerAlaLeuGluVal                          
707580                                                                    
CTCAGGGGCATTGCCTCTGAGACCCACGTGGTCCTCATTCTGAGGGGC645                       
LeuArgGlyIleAlaSerGluThrHisValValLeuIleLeuArgGly                          
859095                                                                    
CCTGAGGGCTTCACTACACATCTGGAGACCACCTTCACAGGGGATGGA693                       
ProGluGlyPheThrThrHisLeuGluThrThrPheThrGlyAspGly                          
100105110115                                                              
ACCCCCAAGACCATCCGGGTGACCCAGCCCCTCGGTCCTCCCACCAAA741                       
ThrProLysThrIleArgValThrGlnProLeuGlyProProThrLys                          
120125130                                                                 
GCCGTCGATCTGTCTCACCAGCCTTCAGCCAGCAAAGACCAGTCATTA789                       
AlaValAspLeuSerHisGlnProSerAlaSerLysAspGlnSerLeu                          
135140145                                                                 
GCAGTAGACAGAGTCACAGGTCTGGGTAATGGCCCTCAGCATGCCCAA837                       
AlaValAspArgValThrGlyLeuGlyAsnGlyProGlnHisAlaGln                          
150155160                                                                 
GGCCATGGGCAGGGAGCTGGCTCAGTCTCCCAAGCTAATGGTGTGGCC885                       
GlyHisGlyGlnGlyAlaGlySerValSerGlnAlaAsnGlyValAla                          
165170175                                                                 
ATTGACCCCACGATGAAAAGCACCAAGGCCAACCTCCAGGACATCGGG933                       
IleAspProThrMetLysSerThrLysAlaAsnLeuGlnAspIleGly                          
180185190195                                                              
GAACATGATGAACTGCTCAAAGAGATAGAACCTGTGCTGAGCATCCTC981                       
GluHisAspGluLeuLeuLysGluIleGluProValLeuSerIleLeu                          
200205210                                                                 
AACAGTGGGAGCAAAGCCACCAACAGAGGGGGACCAGCCAAAGCAGAG1029                      
AsnSerGlySerLysAlaThrAsnArgGlyGlyProAlaLysAlaGlu                          
215220225                                                                 
ATGAAAGACACAGGAATCCAGGTGGACAGAGACCTCGATGGCAAATCG1077                      
MetLysAspThrGlyIleGlnValAspArgAspLeuAspGlyLysSer                          
230235240                                                                 
CACAAAGCTCCGCCCCTGGGCGGGGACAATGACCGCGTCTTCAATGAC1125                      
HisLysAlaProProLeuGlyGlyAspAsnAspArgValPheAsnAsp                          
245250255                                                                 
CTGTGGGGGAAGGACAACGTTCCTGTGATCCTTAACAACCCGTATTCA1173                      
LeuTrpGlyLysAspAsnValProValIleLeuAsnAsnProTyrSer                          
260265270275                                                              
GAGAAGGAACAGTCCCCTACCTCGGGGAAACAGTCTCCCACCAAGAAC1221                      
GluLysGluGlnSerProThrSerGlyLysGlnSerProThrLysAsn                          
280285290                                                                 
GGCAGCCCTTCCAGGTGCCCCCGTTTCCTCAAGGTCAAGAACTGGGAG1269                      
GlySerProSerArgCysProArgPheLeuLysValLysAsnTrpGlu                          
295300305                                                                 
ACGGACGTGGTCCTCACCGACACCCTGCACCTGAAGAGCACACTGGAA1317                      
ThrAspValValLeuThrAspThrLeuHisLeuLysSerThrLeuGlu                          
310315320                                                                 
ACGGGGTGCACAGAGCACATTTGCATGGGCTCGATCATGCTGCCTTCC1365                      
ThrGlyCysThrGluHisIleCysMetGlySerIleMetLeuProSer                          
325330335                                                                 
CAGCACACGCGGAAGCCAGAAGATGTCCGCACAAAGGACCAGCTCTTC1413                      
GlnHisThrArgLysProGluAspValArgThrLysAspGlnLeuPhe                          
340345350355                                                              
CCTCTAGCCAAAGAATTTCTCGACCAATACTACTCATCCATTAAGAGA1461                      
ProLeuAlaLysGluPheLeuAspGlnTyrTyrSerSerIleLysArg                          
360365370                                                                 
TTTGGCTCCAAGGCCCACATGGACAGGCTGGAGGAGGTGAACAAGGAG1509                      
PheGlySerLysAlaHisMetAspArgLeuGluGluValAsnLysGlu                          
375380385                                                                 
ATTGAAAGCACCAGCACCTACCAGCTCAAGGACACCGAGCTCATCTAT1557                      
IleGluSerThrSerThrTyrGlnLeuLysAspThrGluLeuIleTyr                          
390395400                                                                 
GGCGCCAAGCATGCCTGGCGGAACGCCTCTCGATGTGTGGGCAGGATC1605                      
GlyAlaLysHisAlaTrpArgAsnAlaSerArgCysValGlyArgIle                          
405410415                                                                 
CAGTGGTCCAAGCTGCAGGTGTTCGATGCCCGAGACTGCACCACAGCC1653                      
GlnTrpSerLysLeuGlnValPheAspAlaArgAspCysThrThrAla                          
420425430435                                                              
CACGGCATGTTCAACTACATCTGTAACCATGTCAAGTATGCCACCAAC1701                      
HisGlyMetPheAsnTyrIleCysAsnHisValLysTyrAlaThrAsn                          
440445450                                                                 
AAAGGGAATCTCAGGTCGGCCATCACGATATTCCCTCAGAGGACTGAC1749                      
LysGlyAsnLeuArgSerAlaIleThrIlePheProGlnArgThrAsp                          
455460465                                                                 
GGCAAACATGACTTCCGAGTGTGGAACTCGCAGCTCATCCGCTACGCG1797                      
GlyLysHisAspPheArgValTrpAsnSerGlnLeuIleArgTyrAla                          
470475480                                                                 
GGCTACAAGCAGCCAGATGGCTCTACCTTGGGGGATCCAGCCAATGTG1845                      
GlyTyrLysGlnProAspGlySerThrLeuGlyAspProAlaAsnVal                          
485490495                                                                 
CAGTTCACGGAGATCTGTATACAGCAGGGCTGGAAAGCCCCAAGAGGC1893                      
GlnPheThrGluIleCysIleGlnGlnGlyTrpLysAlaProArgGly                          
500505510515                                                              
CGCTTCGACGTGCTGCCTCTCCTGCTTCAGGCCAATGGCAATGACCCT1941                      
ArgPheAspValLeuProLeuLeuLeuGlnAlaAsnGlyAsnAspPro                          
520525530                                                                 
GAGCTCTTCCAGATCCCCCCAGAGCTGGTGCTGGAAGTGCCCATCAGG1989                      
GluLeuPheGlnIleProProGluLeuValLeuGluValProIleArg                          
535540545                                                                 
CACCCCAAGTTCGACTGGTTTAAGGACCTGGGGCTCAAATGGTATGGC2037                      
HisProLysPheAspTrpPheLysAspLeuGlyLeuLysTrpTyrGly                          
550555560                                                                 
CTCCCCGCTGTGTCCAACATGCTGCTGGAGATCGGGGGCCTGGAGTTC2085                      
LeuProAlaValSerAsnMetLeuLeuGluIleGlyGlyLeuGluPhe                          
565570575                                                                 
AGCGCCTGTCCCTTCAGCGGCTGGTACATGGGCACAGAGATCGGCGTC2133                      
SerAlaCysProPheSerGlyTrpTyrMetGlyThrGluIleGlyVal                          
580585590595                                                              
CGTGACTACTGTGACAACTCTCGATACAACATCCTGGAGGAAGTAGCC2181                      
ArgAspTyrCysAspAsnSerArgTyrAsnIleLeuGluGluValAla                          
600605610                                                                 
AAGAAGATGGATTTGGACATGAGGAAGACCTCGTCCCTCTGGAAGGAC2229                      
LysLysMetAspLeuAspMetArgLysThrSerSerLeuTrpLysAsp                          
615620625                                                                 
CAAGCACTGGTGGAGATCAACATTGCTGTTCTATATAGCTTCCAGAGT2277                      
GlnAlaLeuValGluIleAsnIleAlaValLeuTyrSerPheGlnSer                          
630635640                                                                 
GACAAGGTGACCATCGTTGACCACCACTCTGCCACGGAGTCCTTCATC2325                      
AspLysValThrIleValAspHisHisSerAlaThrGluSerPheIle                          
645650655                                                                 
AAACACATGGAGAATGAATACCGCTGCAGAGGGGGCTGCCCCGCCGAC2373                      
LysHisMetGluAsnGluTyrArgCysArgGlyGlyCysProAlaAsp                          
660665670675                                                              
TGGGTGTGGATTGTGCCTCCCATGTCGGGCAGCATCACCCCTGTCTTC2421                      
TrpValTrpIleValProProMetSerGlySerIleThrProValPhe                          
680685690                                                                 
CACCAGGAGATGCTCAACTATAGACTCACCCCGTCCTTTGAATACCAG2469                      
HisGlnGluMetLeuAsnTyrArgLeuThrProSerPheGluTyrGln                          
695700705                                                                 
CCTGATCCATGGAACACCCACGTGTGGAAGGGCACCAACGGGACCCCC2517                      
ProAspProTrpAsnThrHisValTrpLysGlyThrAsnGlyThrPro                          
710715720                                                                 
ACGAAGCGGCGAGCTATCGGCTTTAAGAAATTGGCAGAGGCCGTCAAG2565                      
ThrLysArgArgAlaIleGlyPheLysLysLeuAlaGluAlaValLys                          
725730735                                                                 
TTCTCAGCCAAGCTAATGGGGCAGGCCATGGCCAAGAGGGTCAAGGCG2613                      
PheSerAlaLysLeuMetGlyGlnAlaMetAlaLysArgValLysAla                          
740745750755                                                              
ACCATTCTCTACGCCACAGAGACAGGCAAATCACAAGCCTATGCCAAG2661                      
ThrIleLeuTyrAlaThrGluThrGlyLysSerGlnAlaTyrAlaLys                          
760765770                                                                 
ACCCTGTGTGAGATCTTCAAGCACGCCTTCGATGCCAAGGCAATGTCC2709                      
ThrLeuCysGluIlePheLysHisAlaPheAspAlaLysAlaMetSer                          
775780785                                                                 
ATGGAGGAGTATGACATCGTGCACCTGGAGCACGAAGCCCTGGTCTTG2757                      
MetGluGluTyrAspIleValHisLeuGluHisGluAlaLeuValLeu                          
790795800                                                                 
GTGGTCACCAGCACCTTTGGCAATGGAGACCCCCCTGAGAACGGGGAG2805                      
ValValThrSerThrPheGlyAsnGlyAspProProGluAsnGlyGlu                          
805810815                                                                 
AAATTCGGCTGTGCTTTAATGGAGATGAGGCACCCCAACTCTGTGCAG2853                      
LysPheGlyCysAlaLeuMetGluMetArgHisProAsnSerValGln                          
820825830835                                                              
GAGGAGAGAAAGAGCTACAAGGTCCGATTCAACAGCGTCTCCTCCTAT2901                      
GluGluArgLysSerTyrLysValArgPheAsnSerValSerSerTyr                          
840845850                                                                 
TCTGACTCCCGAAAGTCATCGGGCGACGGACCCGACCTCAGAGACAAC2949                      
SerAspSerArgLysSerSerGlyAspGlyProAspLeuArgAspAsn                          
855860865                                                                 
TTTGAAAGTACTGGACCCCTGGCCAATGTGAGGTTCTCAGTGTTCGGC2997                      
PheGluSerThrGlyProLeuAlaAsnValArgPheSerValPheGly                          
870875880                                                                 
CTCGGCTCTCGGGCGTACCCCCACTTCTGTGCCTTTGGGCATGCGGTG3045                      
LeuGlySerArgAlaTyrProHisPheCysAlaPheGlyHisAlaVal                          
885890895                                                                 
GACACCCTCCTGGAGGAACTGGGAGGGGAGAGGATTCTGAAGATGAGG3093                      
AspThrLeuLeuGluGluLeuGlyGlyGluArgIleLeuLysMetArg                          
900905910915                                                              
GAGGGGGATGAGCTTTGCGGACAGGAAGAAGCTTTCAGGACCTGGGCC3141                      
GluGlyAspGluLeuCysGlyGlnGluGluAlaPheArgThrTrpAla                          
920925930                                                                 
AAGAAAGTCTTCAAGGCAGCCTGTGATGTGTTCTGCGTGGGGGATGAC3189                      
LysLysValPheLysAlaAlaCysAspValPheCysValGlyAspAsp                          
935940945                                                                 
GTCAACATCGAGAAGCCGAACAACTCCCTCATTAGCAATGACCGAAGC3237                      
ValAsnIleGluLysProAsnAsnSerLeuIleSerAsnAspArgSer                          
950955960                                                                 
TGGAAGAGGAACAAGTTCCGCCTCACGTATGTGGCGGAAGCTCCAGAT3285                      
TrpLysArgAsnLysPheArgLeuThrTyrValAlaGluAlaProAsp                          
965970975                                                                 
CTGACCCAAGGTCTTTCCAATGTTCACAAAAAACGAGTCTCGGCTGCT3333                      
LeuThrGlnGlyLeuSerAsnValHisLysLysArgValSerAlaAla                          
980985990995                                                              
CGACTCCTCAGCCGCCAAAACCTGCAAAGCCCTAAGTTCAGCCGATCG3381                      
ArgLeuLeuSerArgGlnAsnLeuGlnSerProLysPheSerArgSer                          
100010051010                                                              
ACCATCTTCGTGCGTCTCCACACCAACGGGAATCAGGAGCTGCAGTAC3429                      
ThrIlePheValArgLeuHisThrAsnGlyAsnGlnGluLeuGlnTyr                          
101510201025                                                              
CAGCCAGGGGACCACCTGGGTGTCTTCCCCGGCAACCACGAGGACCTC3477                      
GlnProGlyAspHisLeuGlyValPheProGlyAsnHisGluAspLeu                          
103010351040                                                              
GTGAATGCACTCATTGAACGGCTGGAGGATGCACCGCCTGCCAACCAC3525                      
ValAsnAlaLeuIleGluArgLeuGluAspAlaProProAlaAsnHis                          
104510501055                                                              
GTGGTGAAGGTGGAGATGCTGGAGGAGAGGAACACTGCTCTGGGTGTC3573                      
ValValLysValGluMetLeuGluGluArgAsnThrAlaLeuGlyVal                          
1060106510701075                                                          
ATCAGTAATTGGAAGGATGAATCTCGCCTCCCACCCTGCACCATCTTC3621                      
IleSerAsnTrpLysAspGluSerArgLeuProProCysThrIlePhe                          
108010851090                                                              
CAGGCCTTCAAGTACTACCTGGACATCACCACGCCGCCCACGCCCCTG3669                      
GlnAlaPheLysTyrTyrLeuAspIleThrThrProProThrProLeu                          
109511001105                                                              
CAGCTGCAGCAGTTCGCCTCTCTGGCCACTAATGAGAAAGAGAAGCAG3717                      
GlnLeuGlnGlnPheAlaSerLeuAlaThrAsnGluLysGluLysGln                          
111011151120                                                              
CGGTTGCTGGTCCTCAGCAAGGGGCTCCAGGAATATGAGGAGTGGAAG3765                      
ArgLeuLeuValLeuSerLysGlyLeuGlnGluTyrGluGluTrpLys                          
112511301135                                                              
TGGGGCAAGAACCCCACAATGGTGGAGGTGCTGGAGGAGTTCCCGTCC3813                      
TrpGlyLysAsnProThrMetValGluValLeuGluGluPheProSer                          
1140114511501155                                                          
ATCCAGATGCCGGCTACACTTCTCCTCACTCAGCTGTCGCTGCTGCAG3861                      
IleGlnMetProAlaThrLeuLeuLeuThrGlnLeuSerLeuLeuGln                          
116011651170                                                              
CCTCGCTACTACTCCATCAGCTCCTCTCCAGACATGTACCCCGACGAG3909                      
ProArgTyrTyrSerIleSerSerSerProAspMetTyrProAspGlu                          
117511801185                                                              
GTGCACCTCACTGTGGCCATCGTCTCCTACCACACCCGAGACGGAGAA3957                      
ValHisLeuThrValAlaIleValSerTyrHisThrArgAspGlyGlu                          
119011951200                                                              
GGACCAGTCCACCACGGGGTGTGCTCCTCCTGGCTCAACAGAATACAG4005                      
GlyProValHisHisGlyValCysSerSerTrpLeuAsnArgIleGln                          
120512101215                                                              
GCTGACGATGTAGTCCCCTGCTTCGTGAGAGGTGCCCCTAGCTTCCAC4053                      
AlaAspAspValValProCysPheValArgGlyAlaProSerPheHis                          
1220122512301235                                                          
CTGCCTCGAAACCCCCAGGTGCCTTGCATCCTGGTTGGCCCAGGCACT4101                      
LeuProArgAsnProGlnValProCysIleLeuValGlyProGlyThr                          
124012451250                                                              
GGCATCGCACCCTTCCGAAGCTTCTGGCAACAGCGACAATTTGACATC4149                      
GlyIleAlaProPheArgSerPheTrpGlnGlnArgGlnPheAspIle                          
125512601265                                                              
CAACACAAAGGAATGAATCCGTGCCCCATGGTTCTGGTCTTCGGGTGT4197                      
GlnHisLysGlyMetAsnProCysProMetValLeuValPheGlyCys                          
127012751280                                                              
CGACAATCCAAGATAGATCATATCTACAGAGAGGAGACCCTGCAGGCT4245                      
ArgGlnSerLysIleAspHisIleTyrArgGluGluThrLeuGlnAla                          
128512901295                                                              
AAGAACAAGGGCGTCTTCAGAGAGCTGTACACTGCCTATTCCCGGGAA4293                      
LysAsnLysGlyValPheArgGluLeuTyrThrAlaTyrSerArgGlu                          
1300130513101315                                                          
CCGGACAGGCCAAAGAAATATGTACAGGACGTGCTGCAGGAACAGCTG4341                      
ProAspArgProLysLysTyrValGlnAspValLeuGlnGluGlnLeu                          
132013251330                                                              
GCTGAGTCTGTGTACCGCGCCCTGAAGGAGCAAGGAGGCCACATTTAT4389                      
AlaGluSerValTyrArgAlaLeuLysGluGlnGlyGlyHisIleTyr                          
133513401345                                                              
GTCTGTGGGGACGTTACCATGGCCGCCGATGTCCTCAAAGCCATCCAG4437                      
ValCysGlyAspValThrMetAlaAlaAspValLeuLysAlaIleGln                          
135013551360                                                              
CGCATAATGACCCAGCAGGGGAAACTCTCAGAGGAGGACGCTGGTGTA4485                      
ArgIleMetThrGlnGlnGlyLysLeuSerGluGluAspAlaGlyVal                          
136513701375                                                              
TTCATCAGCAGGCTGAGGGATGACAACCGGTACCACGAGGACATCTTT4533                      
PheIleSerArgLeuArgAspAspAsnArgTyrHisGluAspIlePhe                          
1380138513901395                                                          
GGAGTCACCCTCAGAACGTATGAAGTGACCAACCGCCTTAGATCTGAG4581                      
GlyValThrLeuArgThrTyrGluValThrAsnArgLeuArgSerGlu                          
140014051410                                                              
TCCATCGCCTTCATCGAAGAGAGCAAAAAAGACGCAGATGAGGTTTTC4629                      
SerIleAlaPheIleGluGluSerLysLysAspAlaAspGluValPhe                          
141514201425                                                              
AGCTCCTAACTGGATCCTCCTGCCCCCGTGCGTGCGATGTGGCGGCTGCCCCAAGT4685              
SerSer                                                                    
143                                                                       
GCCCAAGTAAGGGCGGCCGCAGGTTGACTAAATTCGGACACACACGGCTGAACCGAGTGG4745          
CCCTGCTCTGCCTCTTGTCCTGTTGCTGTGTCCTGGTCCTTCTTCCTGCTCTGGGCTCTC4805          
TCAACCCCACCCCTGGGTTTTCTCCTTGACTCTTGGGCTACGATGCATCACCCTTGTACC4865          
CTGCAGTGGCTCTCACAAAACCGCATCCTCCCCACCCCCACCCGATTGCTGCCAAGGGCA4925          
GGTTGCGGTGCATGGCTGTTGCTCCTGTTGTTGGGGTCTGAAGGTGGCTGGCGCTGGGCC4985          
TCAGGTCACCCTGAACCAGTCCCTTGGCCACTTAAGCCCCCTTCCACCCTCTTTTTATGA5045          
TGGTGTGTTTGT5057                                                          
(2) INFORMATION FOR SEQ ID NO:13:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1429 amino acids                                              
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                  
MetGluGluAsnThrPheGlyValGlnGlnIleGlnProAsnValIle                          
151015                                                                    
SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal                          
202530                                                                    
LysGluArgValSerLysProProValIleIleSerAspLeuIleArg                          
354045                                                                    
GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle                          
505560                                                                    
LeuAlaValAsnAspArgProLeuValAspLeuSerTyrAspSerAla                          
65707580                                                                  
LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle                          
859095                                                                    
LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr                          
100105110                                                                 
GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro                          
115120125                                                                 
ProThrLysAlaValAspLeuSerHisGlnProSerAlaSerLysAsp                          
130135140                                                                 
GlnSerLeuAlaValAspArgValThrGlyLeuGlyAsnGlyProGln                          
145150155160                                                              
HisAlaGlnGlyHisGlyGlnGlyAlaGlySerValSerGlnAlaAsn                          
165170175                                                                 
GlyValAlaIleAspProThrMetLysSerThrLysAlaAsnLeuGln                          
180185190                                                                 
AspIleGlyGluHisAspGluLeuLeuLysGluIleGluProValLeu                          
195200205                                                                 
SerIleLeuAsnSerGlySerLysAlaThrAsnArgGlyGlyProAla                          
210215220                                                                 
LysAlaGluMetLysAspThrGlyIleGlnValAspArgAspLeuAsp                          
225230235240                                                              
GlyLysSerHisLysAlaProProLeuGlyGlyAspAsnAspArgVal                          
245250255                                                                 
PheAsnAspLeuTrpGlyLysAspAsnValProValIleLeuAsnAsn                          
260265270                                                                 
ProTyrSerGluLysGluGlnSerProThrSerGlyLysGlnSerPro                          
275280285                                                                 
ThrLysAsnGlySerProSerArgCysProArgPheLeuLysValLys                          
290295300                                                                 
AsnTrpGluThrAspValValLeuThrAspThrLeuHisLeuLysSer                          
305310315320                                                              
ThrLeuGluThrGlyCysThrGluHisIleCysMetGlySerIleMet                          
325330335                                                                 
LeuProSerGlnHisThrArgLysProGluAspValArgThrLysAsp                          
340345350                                                                 
GlnLeuPheProLeuAlaLysGluPheLeuAspGlnTyrTyrSerSer                          
355360365                                                                 
IleLysArgPheGlySerLysAlaHisMetAspArgLeuGluGluVal                          
370375380                                                                 
AsnLysGluIleGluSerThrSerThrTyrGlnLeuLysAspThrGlu                          
385390395400                                                              
LeuIleTyrGlyAlaLysHisAlaTrpArgAsnAlaSerArgCysVal                          
405410415                                                                 
GlyArgIleGlnTrpSerLysLeuGlnValPheAspAlaArgAspCys                          
420425430                                                                 
ThrThrAlaHisGlyMetPheAsnTyrIleCysAsnHisValLysTyr                          
435440445                                                                 
AlaThrAsnLysGlyAsnLeuArgSerAlaIleThrIlePheProGln                          
450455460                                                                 
ArgThrAspGlyLysHisAspPheArgValTrpAsnSerGlnLeuIle                          
465470475480                                                              
ArgTyrAlaGlyTyrLysGlnProAspGlySerThrLeuGlyAspPro                          
485490495                                                                 
AlaAsnValGlnPheThrGluIleCysIleGlnGlnGlyTrpLysAla                          
500505510                                                                 
ProArgGlyArgPheAspValLeuProLeuLeuLeuGlnAlaAsnGly                          
515520525                                                                 
AsnAspProGluLeuPheGlnIleProProGluLeuValLeuGluVal                          
530535540                                                                 
ProIleArgHisProLysPheAspTrpPheLysAspLeuGlyLeuLys                          
545550555560                                                              
TrpTyrGlyLeuProAlaValSerAsnMetLeuLeuGluIleGlyGly                          
565570575                                                                 
LeuGluPheSerAlaCysProPheSerGlyTrpTyrMetGlyThrGlu                          
580585590                                                                 
IleGlyValArgAspTyrCysAspAsnSerArgTyrAsnIleLeuGlu                          
595600605                                                                 
GluValAlaLysLysMetAspLeuAspMetArgLysThrSerSerLeu                          
610615620                                                                 
TrpLysAspGlnAlaLeuValGluIleAsnIleAlaValLeuTyrSer                          
625630635640                                                              
PheGlnSerAspLysValThrIleValAspHisHisSerAlaThrGlu                          
645650655                                                                 
SerPheIleLysHisMetGluAsnGluTyrArgCysArgGlyGlyCys                          
660665670                                                                 
ProAlaAspTrpValTrpIleValProProMetSerGlySerIleThr                          
675680685                                                                 
ProValPheHisGlnGluMetLeuAsnTyrArgLeuThrProSerPhe                          
690695700                                                                 
GluTyrGlnProAspProTrpAsnThrHisValTrpLysGlyThrAsn                          
705710715720                                                              
GlyThrProThrLysArgArgAlaIleGlyPheLysLysLeuAlaGlu                          
725730735                                                                 
AlaValLysPheSerAlaLysLeuMetGlyGlnAlaMetAlaLysArg                          
740745750                                                                 
ValLysAlaThrIleLeuTyrAlaThrGluThrGlyLysSerGlnAla                          
755760765                                                                 
TyrAlaLysThrLeuCysGluIlePheLysHisAlaPheAspAlaLys                          
770775780                                                                 
AlaMetSerMetGluGluTyrAspIleValHisLeuGluHisGluAla                          
785790795800                                                              
LeuValLeuValValThrSerThrPheGlyAsnGlyAspProProGlu                          
805810815                                                                 
AsnGlyGluLysPheGlyCysAlaLeuMetGluMetArgHisProAsn                          
820825830                                                                 
SerValGlnGluGluArgLysSerTyrLysValArgPheAsnSerVal                          
835840845                                                                 
SerSerTyrSerAspSerArgLysSerSerGlyAspGlyProAspLeu                          
850855860                                                                 
ArgAspAsnPheGluSerThrGlyProLeuAlaAsnValArgPheSer                          
865870875880                                                              
ValPheGlyLeuGlySerArgAlaTyrProHisPheCysAlaPheGly                          
885890895                                                                 
HisAlaValAspThrLeuLeuGluGluLeuGlyGlyGluArgIleLeu                          
900905910                                                                 
LysMetArgGluGlyAspGluLeuCysGlyGlnGluGluAlaPheArg                          
915920925                                                                 
ThrTrpAlaLysLysValPheLysAlaAlaCysAspValPheCysVal                          
930935940                                                                 
GlyAspAspValAsnIleGluLysProAsnAsnSerLeuIleSerAsn                          
945950955960                                                              
AspArgSerTrpLysArgAsnLysPheArgLeuThrTyrValAlaGlu                          
965970975                                                                 
AlaProAspLeuThrGlnGlyLeuSerAsnValHisLysLysArgVal                          
980985990                                                                 
SerAlaAlaArgLeuLeuSerArgGlnAsnLeuGlnSerProLysPhe                          
99510001005                                                               
SerArgSerThrIlePheValArgLeuHisThrAsnGlyAsnGlnGlu                          
101010151020                                                              
LeuGlnTyrGlnProGlyAspHisLeuGlyValPheProGlyAsnHis                          
1025103010351040                                                          
GluAspLeuValAsnAlaLeuIleGluArgLeuGluAspAlaProPro                          
104510501055                                                              
AlaAsnHisValValLysValGluMetLeuGluGluArgAsnThrAla                          
106010651070                                                              
LeuGlyValIleSerAsnTrpLysAspGluSerArgLeuProProCys                          
107510801085                                                              
ThrIlePheGlnAlaPheLysTyrTyrLeuAspIleThrThrProPro                          
109010951100                                                              
ThrProLeuGlnLeuGlnGlnPheAlaSerLeuAlaThrAsnGluLys                          
1105111011151120                                                          
GluLysGlnArgLeuLeuValLeuSerLysGlyLeuGlnGluTyrGlu                          
112511301135                                                              
GluTrpLysTrpGlyLysAsnProThrMetValGluValLeuGluGlu                          
114011451150                                                              
PheProSerIleGlnMetProAlaThrLeuLeuLeuThrGlnLeuSer                          
115511601165                                                              
LeuLeuGlnProArgTyrTyrSerIleSerSerSerProAspMetTyr                          
117011751180                                                              
ProAspGluValHisLeuThrValAlaIleValSerTyrHisThrArg                          
1185119011951200                                                          
AspGlyGluGlyProValHisHisGlyValCysSerSerTrpLeuAsn                          
120512101215                                                              
ArgIleGlnAlaAspAspValValProCysPheValArgGlyAlaPro                          
122012251230                                                              
SerPheHisLeuProArgAsnProGlnValProCysIleLeuValGly                          
123512401245                                                              
ProGlyThrGlyIleAlaProPheArgSerPheTrpGlnGlnArgGln                          
125012551260                                                              
PheAspIleGlnHisLysGlyMetAsnProCysProMetValLeuVal                          
1265127012751280                                                          
PheGlyCysArgGlnSerLysIleAspHisIleTyrArgGluGluThr                          
128512901295                                                              
LeuGlnAlaLysAsnLysGlyValPheArgGluLeuTyrThrAlaTyr                          
130013051310                                                              
SerArgGluProAspArgProLysLysTyrValGlnAspValLeuGln                          
131513201325                                                              
GluGlnLeuAlaGluSerValTyrArgAlaLeuLysGluGlnGlyGly                          
133013351340                                                              
HisIleTyrValCysGlyAspValThrMetAlaAlaAspValLeuLys                          
1345135013551360                                                          
AlaIleGlnArgIleMetThrGlnGlnGlyLysLeuSerGluGluAsp                          
136513701375                                                              
AlaGlyValPheIleSerArgLeuArgAspAspAsnArgTyrHisGlu                          
138013851390                                                              
AspIlePheGlyValThrLeuArgThrTyrGluValThrAsnArgLeu                          
139514001405                                                              
ArgSerGluSerIleAlaPheIleGluGluSerLysLysAspAlaAsp                          
141014151420                                                              
GluValPheSerSer                                                           
1425                                                                      
(2) INFORMATION FOR SEQ ID NO:14:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 5086 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: human bcl-2 cDNA                                  
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 1459..2178                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                  
GCGCCCGCCCCTCCGCGCCGCCTGCCCGCCCGCCCGCCGCGCTCCCGCCCGCCGCTCTCC60            
GTGGCCCCGCCGCGCTGCCGCCGCCGCCGCTGCCAGCGAAGGTGCCGGGGCTCCGGGCCC120           
TCCCTGCCGGCGGCCGTCAGCGCTCGGAGCGAACTGCGCGACGGGAGGTCCGGGAGGCGA180           
CCGTAGTCGCGCCGCCGCGCAGGACCAGGAGGAGGAGAAAGGGTGCGCAGCCCGGAGGCG240           
GGGTGCGCCGGTGGGGTGCAGCGGAAGAGGGGGTCCAGGGGGGAGAACTTCGTAGCAGTC300           
ATCCTTTTTAGGAAAAGAGGGAAAAAATAAAACCCTCCCCCACCACCTCCTTCTCCCCAC360           
CCCTCGCCGCACCACACACAGCGCGGGCTTCTAGCGCTCGGCACCGGCGGGCCAGGCGCG420           
TCCTGCCTTCATTTATCCAGCAGCTTTTCGGAAAATGCATTTGCTGTTCGGAGTTTAATC480           
AGAAGACGATTCCTGCCTCCGTCCCCGGCTCCTTCATCGTCCCATCTCCCCTGTCTCTCT540           
CCTGGGGAGGCGTGAAGCGGTCCCGTGGATAGAGATTCATGCCTGTGTCCGCGCGTGTGT600           
GCGCGCGTATAAATTGCCGAGAAGGGGAAAACATCACAGGACTTCTGCGAATACCGGACT660           
GAAAATTGTAATTCATCTGCCGCCGCCGCTGCCAAAAAAAAACTCGAGCTCTTGAGATCT720           
CCGGTTGGGATTCCTGCGGATTGACATTTCTGTGAAGCAGAAGTCTGGGAATCGATCTGG780           
AAATCCTCCTAATTTTTACTCCCTCTCCCCCCGACTCCTGATTCATTGGGAAGTTTCAAA840           
TCAGCTATAACTGGAGAGTGCTGAAGATTGATGGGATCGTTGCCTTATGCATTTGTTTTG900           
GTTTTACAAAAAGGAAACTTGACAGAGGATCATGCTGTACTTAAAAAATACAAGTAAGTC960           
TCGCACAGGAAATTGGTTTAATGTAACTTTCAATGGAAACCTTTGAGATTTTTTACTTAA1020          
AGTGCATTCGAGTAAATTTAATTTCCAGGCAGCTTAATACATTGTTTTTAGCCGTGTTAC1080          
TTGTAGTGTGTATGCCCTGCTTTCACTCAGTGTGTACAGGGAAACGCACCTGATTTTTTA1140          
CTTATTAGTTTGTTTTTTCTTTAACCTTTCAGCATCACAGAGGAAGTAGACTGATATTAA1200          
CAATACTTACTAATAATAACGTGCCTCATGAAATAAAGATCCGAAAGGAATTGGAATAAA1260          
AATTTCCTGCGTCTCATGCCAAGAGGGAAACACCAGAATCAAGTGTTCCGCGTGATTGAA1320          
GACACCCCCTCGTCCAAGAATGCAAAGCACATCCAATAAAATAGCTGGATTATAACTCCT1380          
CTTCTTTCTCTGGGGGCCGTGGGGTGGGAGCTGGGGCGAGAGGTGCCGTTGGCCCCCGTT1440          
GCTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGAACGGGGTACGACAAC1491                   
MetAlaHisAlaGlyArgThrGlyTyrAspAsn                                         
1510                                                                      
CGGGAGATAGTGATGAAGTACATCCATTATAAGCTGTCGCAGAGGGGC1539                      
ArgGluIleValMetLysTyrIleHisTyrLysLeuSerGlnArgGly                          
152025                                                                    
TACGAGTGGGATGCGGGAGATGTGGGCGCCGCGCCCCCGGGGGCCGCC1587                      
TyrGluTrpAspAlaGlyAspValGlyAlaAlaProProGlyAlaAla                          
303540                                                                    
CCCGCACCGGGCATCTTCTCCTCCCAGCCCGGGCACACGCCCCATCCA1635                      
ProAlaProGlyIlePheSerSerGlnProGlyHisThrProHisPro                          
455055                                                                    
GCCGCATCCCGCGACCCGGTCGCCAGGACCTCGCCGCTGCAGACCCCG1683                      
AlaAlaSerArgAspProValAlaArgThrSerProLeuGlnThrPro                          
60657075                                                                  
GCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCACCT1731                      
AlaAlaProGlyAlaAlaAlaGlyProAlaLeuSerProValProPro                          
808590                                                                    
GTGGTCCACCTGGCCCTCCGCCAAGCCGGCGACGACTTCTCCCGCCGC1779                      
ValValHisLeuAlaLeuArgGlnAlaGlyAspAspPheSerArgArg                          
95100105                                                                  
TACCGCGGCGACTTCGCCGAGATGTCCAGCCAGCTGCACCTGACGCCC1827                      
TyrArgGlyAspPheAlaGluMetSerSerGlnLeuHisLeuThrPro                          
110115120                                                                 
TTCACCGCGCGGGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGG1875                      
PheThrAlaArgGlyArgPheAlaThrValValGluGluLeuPheArg                          
125130135                                                                 
GACGGGGTGAACTGGGGGAGGATTGTGGCCTTCTTTGAGTTCGGTGGG1923                      
AspGlyValAsnTrpGlyArgIleValAlaPhePheGluPheGlyGly                          
140145150155                                                              
GTCATGTGTGTGGAGAGCGTCAACCGGGAGATGTCGCCCCTGGTGGAC1971                      
ValMetCysValGluSerValAsnArgGluMetSerProLeuValAsp                          
160165170                                                                 
AACATCGCCCTGTGGATGACTGAGTACCTGAACCGGCACCTGCACACC2019                      
AsnIleAlaLeuTrpMetThrGluTyrLeuAsnArgHisLeuHisThr                          
175180185                                                                 
TGGATCCAGGATAACGGAGGCTGGGATGCCTTTGTGGAACTGTACGGC2067                      
TrpIleGlnAspAsnGlyGlyTrpAspAlaPheValGluLeuTyrGly                          
190195200                                                                 
CCCAGCATGCGGCCTCTGTTTGATTTCTCCTGGCTGTCTCTGAAGACT2115                      
ProSerMetArgProLeuPheAspPheSerTrpLeuSerLeuLysThr                          
205210215                                                                 
CTGCTCAGTTTGGCCCTGGTGGGAGCTTGCATCACCCTGGGTGCCTAT2163                      
LeuLeuSerLeuAlaLeuValGlyAlaCysIleThrLeuGlyAlaTyr                          
220225230235                                                              
CTGAGCCACAAGTGAAGTCAACATGCCTGCCCCAAACAAATATGCAAAAGGT2215                  
LeuSerHisLys                                                              
240                                                                       
TCACTAAAGCAGTAGAAATAATATGCATTGTCAGTGATGTACCATGAAACAAAGCTGCAG2275          
GCTGTTTAAGAAAAAATAACACACATATAAACATCACACACACAGACAGACACACACACA2335          
CACAACAATTAACAGTCTTCAGGCAAAACGTCGAATCAGCTATTTACTGCCAAAGGGAAA2395          
TATCATTTATTTTTTACATTATTAAGAAAAAAGATTTATTTATTTAAGACAGTCCCATCA2455          
AAACTCCGTCTTTGGAAATCCGACCACTAATTGCCAAACACCGCTTCGTGTGGCTCCACC2515          
TGGATGTTCTGTGCCTGTAAACATAGATTCGCTTTCCATGTTGTTGGCCGGATCACCATC2575          
TGAAGAGCAGACGGATGGAAAAAGGACCTGATCATTGGGGAAGCTGGCTTTCTGGCTGCT2635          
GGAGGCTGGGGAGAAGGTGTTCATTCACTTGCATTTCTTTGCCCTGGGGGCGTGATATTA2695          
ACAGAGGGAGGGTTCCCGTGGGGGGAAGTCCATGCCTCCCTGGCCTGAAGAAGAGACTCT2755          
TTGCATATGACTCACATGATGCATACCTGGTGGGAGGAAAAGAGTTGGGAACTTCAGATG2815          
GACCTAGTACCCACTGAGATTTCCACGCCGAAGGACAGCGATGGGAAAAATGCCCTTAAA2875          
TCATAGGAAAGTATTTTTTTAAGCTACCAATTGTGCCGAGAAAAGCATTTTAGCAATTTA2935          
TACAATATCATCCAGTACCTTAAACCCTGATTGTGTATATTCATATATTTTGGATACGCA2995          
CCCCCCAACTCCCAATACTGGCTCTGTCTGAGTAAGAAACAGAATCCTCTGGAACTTGAG3055          
GAAGTGAACATTTCGGTGACTTCCGATCAGGAAGGCTAGAGTTACCCAGAGCATCAGGCC3115          
GCCACAAGTGCCTGCTTTTAGGAGACCGAAGTCCGCAGAACCTACCTGTGTCCCAGCTTG3175          
GAGGCCTGGTCCTGGAACTGAGCCGGGCCCTCACTGGCCTCCTCCAGGGATGATCAACAG3235          
GGTAGTGTGGTCTCCGAATGTCTGGAAGCTGATGGATGGAGCTCAGAATTCCACTGTCAA3295          
GAAAGAGCAGTAGAGGGGTGTGGCTGGGCCTGTCACCCTGGGGCCCTCCAGGTAGGCCCG3355          
TTTTCACGTGGAGCATAGGAGCCACGACCCTTCTTAAGACATGTATCACTGTAGAGGGAA3415          
GGAACAGAGGCCCTGGGCCTTCCTATCAGAAGGACATGGTGAAGGCTGGGAACGTGAGGA3475          
GAGGCAATGGCCACGGCCCATTTTGGCTGTAGCACATGGCACGTTGGCTGTGTGGCCTTG3535          
GCCACCTGTGAGTTTAAAGCAAGGCTTTAAATGACTTTGGAGAGGGTCACAAATCCTAAA3595          
AGAAGCATTGAAGTGAGGTGTCATGGATTAATTGACCCCTGTCTATGGAATTACATGTAA3655          
AACATTATCTTGTCACTGTAGTTTGGTTTTATTTGAAAACCTGACAAAAAAAAAGTTCCA3715          
GGTGTGGAATATGGGGGTTATCTGTACATCCTGGGGCATTAAAAAAAAATCAATGGTGGG3775          
GAACTATAAAGAAGTAACAAAAGAAGTGACATCTTCAGCAAATAAACTAGGAAATTTTTT3835          
TTTCTTCCAGTTTAGAATCAGCCTTGAAACATTGATGGAATAACTCTGTGGCATTATTGC3895          
ATTATATACCATTTATCTGTATTAACTTTGGAATGTACTCTGTTCAATGTTTAATGCTGT3955          
GGTTGATATTTCGAAAGCTGCTTTAAAAAAATACATGCATCTCAGCGTTTTTTTGTTTTT4015          
AATTGTATTTAGTTATGGCCTATACACTATTTGTGAGCAAAGGTGATCGTTTTCTGTTTG4075          
AGATTTTTATCTCTTGATTCTTCAAAAGCATTCTGAGAAGGTGAGATAAGCCCTGAGTCT4135          
CAGCTACCTAAGAAAAACCTGGATGTCACTGGCCACTGAGGAGCTTTGTTTCAACCAAGT4195          
CATGTGCATTTCCACGTCAACAGAATTGTTTATTGTGACAGTTATATCTGTTGTCCCTTT4255          
GACCTTGTTTCTTGAAGGTTTCCTCGTCCCTGGGCAATTCCGCATTTAATTCATGGTATT4315          
CAGGATTACATGCATGTTTGGTTAAACCCATGAGATTCATTCAGTTAAAAATCCAGATGG4375          
CGAATGACCAGCAGATTCAAATCTATGGTGGTTTGACCTTTAGAGAGTTGCTTTACGTGG4435          
CCTGTTTCAACACAGACCCACCCAGAGCCCTCCTGCCCTCCTTCCGCGGGGGCTTTCTCA4495          
TGGCTGTCCTTCAGGGTCTTCCTGAAATGCAGTGGTCGTTACGCTCCACCAAGAAAGCAG4555          
GAAACCTGTGGTATGAAGCCAGACCTCCCCGGCGGGCCTCAGGGAACAGAATGATCAGAC4615          
CTTTGAATGATTCTAATTTTTAAGCAAAATATTATTTTATGAAAGGTTTACATTGTCAAA4675          
GTGATGAATATGGAATATCCAATCCTGTGCTGCTATCCTGCCAAAATCATTTTAATGGAG4735          
TCAGTTTGCAGTATGCTCCACGTGGTAAGATCCTCCAAGCTGCTTTAGAAGTAACAATGA4795          
AGAACGTGGACGTTTTTAATATAAAGCCTGTTTTGTCTTTTGTTGTTGTTCAAACGGGAT4855          
TCACAGAGTATTTGAAAAATGTATATATATTAAGAGGTCACGGGGGCTAATTGCTAGCTG4915          
GCTGCCTTTTGCTGTGGGGTTTTGTTACCTGGTTTTAATAACAGTAAATGTGCCCAGCCT4975          
CTTGGCCCCAGAACTGTACAGTATTGTGGCTGCACTTGCTCTAAGAGTAGTTGATGTTGC5035          
ATTTTCCTTATTGTTAAAAACATGTTAGAAGCAATGAATGTATATAAAAGC5086                   
(2) INFORMATION FOR SEQ ID NO:15:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 239 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                  
MetAlaHisAlaGlyArgThrGlyTyrAspAsnArgGluIleValMet                          
151015                                                                    
LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla                          
202530                                                                    
GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyIle                          
354045                                                                    
PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp                          
505560                                                                    
ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla                          
65707580                                                                  
AlaAlaGlyProAlaLeuSerProValProProValValHisLeuAla                          
859095                                                                    
LeuArgGlnAlaGlyAspAspPheSerArgArgTyrArgGlyAspPhe                          
100105110                                                                 
AlaGluMetSerSerGlnLeuHisLeuThrProPheThrAlaArgGly                          
115120125                                                                 
ArgPheAlaThrValValGluGluLeuPheArgAspGlyValAsnTrp                          
130135140                                                                 
GlyArgIleValAlaPhePheGluPheGlyGlyValMetCysValGlu                          
145150155160                                                              
SerValAsnArgGluMetSerProLeuValAspAsnIleAlaLeuTrp                          
165170175                                                                 
MetThrGluTyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsn                          
180185190                                                                 
GlyGlyTrpAspAlaPheValGluLeuTyrGlyProSerMetArgPro                          
195200205                                                                 
LeuPheAspPheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAla                          
210215220                                                                 
LeuValGlyAlaCysIleThrLeuGlyAlaTyrLeuSerHisLys                             
225230235                                                                 
(2) INFORMATION FOR SEQ ID NO:16:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1846 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: bcl-2 fusion gene; Seto, et al.,                  
EMBO J 7:123 (1988)                                                       
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 887..1606                                                   
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                  
ACCACCTCCTTCTCCCCACCCCTCGCCGCACCACACACAGCGCGGGCTTCTGGCGCTCGG60            
CACCGGCGGGCCAGGCGCGTCCTGTCTTCATTTATCCAGCAGCTTTTCGGAAAATCCATT120           
TGGTGTTCGGAGTTTAATCAGAAGAGGATTCCTGCCTCCGTCCCCGGCTCCTTCATCGTC180           
CCCTCTCCCCTGTCTCTCTCCTGGGGAGGCGTGAAGAGAGATTCATGCCTGTGCCCGCGC240           
GTGTGTGCGCGCGTATAAATTGCCGAGAAGGGGAAAACATCACAGGACTTCTGCGAATAC300           
CGGACTGAAAATTGTAGCTCATCTGCCGCCGCCGCTGCCTTTTTTTTTTCTCGAGCTCTT360           
GAGATCTCCGGTTGGGACTCCTGCGGATTGACATTTCTGTGAAGCAGAAGTCTGGGAATC420           
GATCTGGAAATCCTCCTAATTTTTACTCCCTCTCCCCCCGACTCCTGATTCATTGGGAAG480           
TTTCAAATCAGCTATAACTGGAGAGAGCTGAAGATTGATGGGATCGTTGCCTTATGCCTT540           
TGTTTTGGTTTTACAAAAAGGAAACTTGACAGAGGATCATGCTATACTTAAAAAATACAA600           
CATCGCAGAGGAAGTAGACTCATATTAAAAATACTTACTAATAATAACGTGCCTCATGAA660           
GTAAAGATCCGAAAGGAATTGGAATAAAACTTTCCTGCATCTCAAGCCAAGGGGGAAACA720           
CCAGAATCAAGTGTTCCGCGTGATTGAAGACACCCCCTCGTCCAAGAATGCAAAGCACAT780           
CCAATAAAAGAGCTGGATTATAACTCCTCTTCTTTCTCTGGGGGCCGTGGGGTAGGGGCT840           
GGGGCGAGAGGTGCCGTTGGCCCCCGTTGCTTTTCCTCTGGGAGGGATGGCGCAC895                
MetAlaHis                                                                 
1                                                                         
GCTGGGAGAAGTGGTTACGATAACCGGGAGATAGTGATGAAGTACATC943                       
AlaGlyArgSerGlyTyrAspAsnArgGluIleValMetLysTyrIle                          
51015                                                                     
CATTATAAGCTGTCGCAGAGGGGCTACGAGTGGGATGCGGGAGATGTG991                       
HisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAlaGlyAspVal                          
20253035                                                                  
GGCGCCGCGCCCCCGGGGGCCGCCCCCGCACCGGGCTTCTTCTCCTCC1039                      
GlyAlaAlaProProGlyAlaAlaProAlaProGlyPhePheSerSer                          
404550                                                                    
CAGCCCGGGCACACGCCCCATCCAGCCGCATCCCGGGACCCGGTCGCC1087                      
GlnProGlyHisThrProHisProAlaAlaSerArgAspProValAla                          
556065                                                                    
AGGACCTCGCCACTACAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGG1135                      
ArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAlaGly                          
707580                                                                    
CCTGCGCTCAGCCCGGTGCCACCTGTGGTCCACCTGACCCTCCGCCAG1183                      
ProAlaLeuSerProValProProValValHisLeuThrLeuArgGln                          
859095                                                                    
GCCGGCGACGACTTCTCCCGCCGCTACCGCCGCGACTTCGCCGAGATG1231                      
AlaGlyAspAspPheSerArgArgTyrArgArgAspPheAlaGluMet                          
100105110115                                                              
TCCAGCCAGCTGCACCTGACGCCCTTCACCGCGCGGGGATGCTTTGCC1279                      
SerSerGlnLeuHisLeuThrProPheThrAlaArgGlyCysPheAla                          
120125130                                                                 
ACGGTGGTGGAGGAGCTCTTCAGGGACGGGGTGAACTGGGGGAGGATT1327                      
ThrValValGluGluLeuPheArgAspGlyValAsnTrpGlyArgIle                          
135140145                                                                 
GTGGCCTTCTTTGAGTTCGGTGGGGTCATGTGTGTGGAGAGCGTCAAC1375                      
ValAlaPhePheGluPheGlyGlyValMetCysValGluSerValAsn                          
150155160                                                                 
CGGGAGATGTCGCCCCTGGTGGACAACATCGCCCTGTGGATGACTGAG1423                      
ArgGluMetSerProLeuValAspAsnIleAlaLeuTrpMetThrGlu                          
165170175                                                                 
TACCTGAACCGGCACCTGCACACCTGGATCCAGGATAACGGAGGCTGG1471                      
TyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsnGlyGlyTrp                          
180185190195                                                              
GATGCCTTTGTGGAACTGTACGGCCCCAGCATGCGGCCTCTGTTTGAT1519                      
AspAlaPheValGluLeuTyrGlyProSerMetArgProLeuPheAsp                          
200205210                                                                 
TTCTCCTGGCTGTCTCTGAAGACTCTGCTCAGTTTGGCCCTGGTGGGA1567                      
PheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAlaLeuValGly                          
215220225                                                                 
GCTTGCATCACCCTGGGTGCCTATCTGGGCCACAAGTGAAGTCAAC1613                        
AlaCysIleThrLeuGlyAlaTyrLeuGlyHisLys                                      
230235240                                                                 
ATGCCTGCCCCAAACAAATATGCAAAAGGTTCACTAAAGCAGTAGAAATAATATGCATTG1673          
TCAGTGATGTACCATGAAACAAAGCTGCAGGCTGTTTAAGAAAAAATAACACACATATAA1733          
ACATCACACACACAGACAGACACACACACACACAACAATTAACAGTCTTCAGGCAAAACG1793          
TCGAATCAGCTATTTACTGCCAAAGGGAAATATCATTTATTTTTTACATTATT1846                 
(2) INFORMATION FOR SEQ ID NO:17:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 239 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                  
MetAlaHisAlaGlyArgSerGlyTyrAspAsnArgGluIleValMet                          
151015                                                                    
LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla                          
202530                                                                    
GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyPhe                          
354045                                                                    
PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp                          
505560                                                                    
ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla                          
65707580                                                                  
AlaAlaGlyProAlaLeuSerProValProProValValHisLeuThr                          
859095                                                                    
LeuArgGlnAlaGlyAspAspPheSerArgArgTyrArgArgAspPhe                          
100105110                                                                 
AlaGluMetSerSerGlnLeuHisLeuThrProPheThrAlaArgGly                          
115120125                                                                 
CysPheAlaThrValValGluGluLeuPheArgAspGlyValAsnTrp                          
130135140                                                                 
GlyArgIleValAlaPhePheGluPheGlyGlyValMetCysValGlu                          
145150155160                                                              
SerValAsnArgGluMetSerProLeuValAspAsnIleAlaLeuTrp                          
165170175                                                                 
MetThrGluTyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsn                          
180185190                                                                 
GlyGlyTrpAspAlaPheValGluLeuTyrGlyProSerMetArgPro                          
195200205                                                                 
LeuPheAspPheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAla                          
210215220                                                                 
LeuValGlyAlaCysIleThrLeuGlyAlaTyrLeuGlyHisLys                             
225230235                                                                 
(2) INFORMATION FOR SEQ ID NO:18:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 4353 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: Human NOS-1 gene, Fujisawa, et al,                
J. Neurochem 63:140 1994                                                  
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 1..4305                                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                  
ATGGAGGATCACATGTTCGGTGTTCAGCAAATCCAGCCCAATGTCATT48                        
MetGluAspHisMetPheGlyValGlnGlnIleGlnProAsnValIle                          
151015                                                                    
TCTGTTCGTCTCTTCAAGCGCAAAGTTGGGGGCCTGGGATTTCTGGTG96                        
SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal                          
202530                                                                    
AAGGAGCGGGTCAGTAAGCCGCCCGTGATCATCTCTGACCTGATTCGT144                       
LysGluArgValSerLysProProValIleIleSerAspLeuIleArg                          
354045                                                                    
GGGGGCGCCGCAGAGCAGAGTGGCCTCATCCAGGCCGGAGACATCATT192                       
GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle                          
505560                                                                    
CTTGCGGTCAACGGCCGGCCCTTGGTGGACCTGAGCTATGACAGCGCC240                       
LeuAlaValAsnGlyArgProLeuValAspLeuSerTyrAspSerAla                          
65707580                                                                  
CTGGAGGTACTCAGAGGCATTGCCTCTGAGACCCACGTGGTCCTCATT288                       
LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle                          
859095                                                                    
CTGAGGGGCCCTGAAGGTTTCACCACGCACCTGGAGACCACCTTTACA336                       
LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr                          
100105110                                                                 
GGTGATGGGACCCCCAAGACCATCCGGGTGACACAGCCCCTGGGTCCC384                       
GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro                          
115120125                                                                 
CCCACCAAAGCCGTGGATCTGTCCCACCAGCCACCGGCCGGCAAAGAA432                       
ProThrLysAlaValAspLeuSerHisGlnProProAlaGlyLysGlu                          
130135140                                                                 
CAGCCCCTGGCAGTGGATGGGGCCTCGGGTCCCGGGAATGGGCCTCAG480                       
GlnProLeuAlaValAspGlyAlaSerGlyProGlyAsnGlyProGln                          
145150155160                                                              
CATGCCTACGATGATGGGCAGGAGGCTGGCTCACTCCCCCATGCCAAC528                       
HisAlaTyrAspAspGlyGlnGluAlaGlySerLeuProHisAlaAsn                          
165170175                                                                 
GGCCTGGCCCCCAGGCCCCCAGGCCAGGACCCCGCGAAGAAAGCAACC576                       
GlyLeuAlaProArgProProGlyGlnAspProAlaLysLysAlaThr                          
180185190                                                                 
AGAGTCAGCCTCCAAGGCAGAGGGGAGAACAATGAACTGCTCAAGGAG624                       
ArgValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeuLysGlu                          
195200205                                                                 
ATAGAGCCTGTGCTGAGCCTTCTCACCAGTGGGAGCAGAGGGGTCAAG672                       
IleGluProValLeuSerLeuLeuThrSerGlySerArgGlyValLys                          
210215220                                                                 
GGAGGGGCACCTGCCAAGGCAGAGATGAAAGATATGGGAATCCAGGTG720                       
GlyGlyAlaProAlaLysAlaGluMetLysAspMetGlyIleGlnVal                          
225230235240                                                              
GACAGAGATTTGGACGGCAAGTCACACAAACCTCTGCCCCTCGGCGTG768                       
AspArgAspLeuAspGlyLysSerHisLysProLeuProLeuGlyVal                          
245250255                                                                 
GAGAACGACCGAGTCTTCAATGACCTATGGGGGAAGGGCAATGTGCCT816                       
GluAsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsnValPro                          
260265270                                                                 
GTCGTCCTCAACAACCCATATTCAGAGAAGGAGCAGCCCCCCACCTCA864                       
ValValLeuAsnAsnProTyrSerGluLysGluGlnProProThrSer                          
275280285                                                                 
GGAAAACAGTCCCCCACAAAGAATGGCAGCCCCTCCAAGTGTCCACGC912                       
GlyLysGlnSerProThrLysAsnGlySerProSerLysCysProArg                          
290295300                                                                 
TTCCTCAAGGTCAAGAACTGGGAGACTGAGGTGGTTCTCACTGACACC960                       
PheLeuLysValLysAsnTrpGluThrGluValValLeuThrAspThr                          
305310315320                                                              
CTCCACCTTAAGAGCACATTGGAAACGGGATGCACTGAGTACATCTGC1008                      
LeuHisLeuLysSerThrLeuGluThrGlyCysThrGluTyrIleCys                          
325330335                                                                 
ATGGGCTCCATCATGCATCCTTCTCAGCATGCAAGGAGGCCTGAAGAC1056                      
MetGlySerIleMetHisProSerGlnHisAlaArgArgProGluAsp                          
340345350                                                                 
GTCCGCACAAAAGGACAGCTCTTCCCTCTCGCCAAAGAGTTTATTGAT1104                      
ValArgThrLysGlyGlnLeuPheProLeuAlaLysGluPheIleAsp                          
355360365                                                                 
CAATACTATTCATCAATTAAAAGATTTGGCTCCAAAGCCCACATGGAA1152                      
GlnTyrTyrSerSerIleLysArgPheGlySerLysAlaHisMetGlu                          
370375380                                                                 
AGGCTGGAAGAGGTGAACAAAGAGATCGACACCACTAGCACTTACCAG1200                      
ArgLeuGluGluValAsnLysGluIleAspThrThrSerThrTyrGln                          
385390395400                                                              
CTCAAGGACACAGAGCTCATCTATGGGGCCAAGCACGCCTGGCGGAAT1248                      
LeuLysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrpArgAsn                          
405410415                                                                 
GCCTCGCGCTGTGTGGGCAGGATCCAGTGGTCCAAGCTGCAGGTATTC1296                      
AlaSerArgCysValGlyArgIleGlnTrpSerLysLeuGlnValPhe                          
420425430                                                                 
GATGCCCGTGACTGCACCACGGCCCACGGGATGTTCAACTACATCTGT1344                      
AspAlaArgAspCysThrThrAlaHisGlyMetPheAsnTyrIleCys                          
435440445                                                                 
AACCATGTCAAGTATGCCACCAACAAAGGGAACCTCAGGTCTGCCATC1392                      
AsnHisValLysTyrAlaThrAsnLysGlyAsnLeuArgSerAlaIle                          
450455460                                                                 
ACCATATTCCCCCAGAGGACAGACGGCAAGCACGACTTCCGAGTCTGG1440                      
ThrIlePheProGlnArgThrAspGlyLysHisAspPheArgValTrp                          
465470475480                                                              
AACTCCCAGCTCATCCGCTACGCTGGCTACAAGCAGCCTGACGGCTCC1488                      
AsnSerGlnLeuIleArgTyrAlaGlyTyrLysGlnProAspGlySer                          
485490495                                                                 
ACCCTGGGGGACCCAGCCAATGTGCAGTTCACAGAGATATGCATACAG1536                      
ThrLeuGlyAspProAlaAsnValGlnPheThrGluIleCysIleGln                          
500505510                                                                 
CAGGGCTGGAAACCGCCTAGAGGCCGCTTCGATGTCCTGCCGCTCCTG1584                      
GlnGlyTrpLysProProArgGlyArgPheAspValLeuProLeuLeu                          
515520525                                                                 
CTTCAGGCCAACGGCAATGACCCTGAGCTCTTCCAGATTCCTCCAGAG1632                      
LeuGlnAlaAsnGlyAsnAspProGluLeuPheGlnIleProProGlu                          
530535540                                                                 
CTGGTGTTGGAAGTTCCCATCAGGCACCCCAAGTTTGAGTGGTTCAAG1680                      
LeuValLeuGluValProIleArgHisProLysPheGluTrpPheLys                          
545550555560                                                              
GACCTGGGGCTGAAGTGGTACGGCCTCCCCGCCGTGTCCAACATGCTC1728                      
AspLeuGlyLeuLysTrpTyrGlyLeuProAlaValSerAsnMetLeu                          
565570575                                                                 
CTAGAGATTGGCGGCCTGGAGTTCAGCGCCTGTCCCTTCAGTGGCTGG1776                      
LeuGluIleGlyGlyLeuGluPheSerAlaCysProPheSerGlyTrp                          
580585590                                                                 
TACATGGGCACAGAGATTGGTGTCCGCGACTACTGTGACAACTCCCGC1824                      
TyrMetGlyThrGluIleGlyValArgAspTyrCysAspAsnSerArg                          
595600605                                                                 
TACAATATCCTGGAGGAAGTGGCCAAGAAGATGAACTTAGACATGAGG1872                      
TyrAsnIleLeuGluGluValAlaLysLysMetAsnLeuAspMetArg                          
610615620                                                                 
AAGACGTCCTCCCTGTGGAAGGACCAGGCGCTGGTGGAGATCAATATC1920                      
LysThrSerSerLeuTrpLysAspGlnAlaLeuValGluIleAsnIle                          
625630635640                                                              
GCGGTTCTCTATAGCTTCCAGAGTGACAAAGTGACCATTGTTGACCAT1968                      
AlaValLeuTyrSerPheGlnSerAspLysValThrIleValAspHis                          
645650655                                                                 
CACTCCGCCACCGAGTCCTTCATTAAGCACATGGAGAATGAGTACCGC2016                      
HisSerAlaThrGluSerPheIleLysHisMetGluAsnGluTyrArg                          
660665670                                                                 
TGCCGGGGGGGCTGCCCTGCCGACTGGGTGTGGATCGTGCCCCCCATG2064                      
CysArgGlyGlyCysProAlaAspTrpValTrpIleValProProMet                          
675680685                                                                 
TCCGGAAGCATCACCCCTGTGTTCCACCAGGAGATGCTCAACTACCGG2112                      
SerGlySerIleThrProValPheHisGlnGluMetLeuAsnTyrArg                          
690695700                                                                 
CTCACCCCCTCCTTCGAATACCAGCCTGATCCCTGGAACACGCATGTC2160                      
LeuThrProSerPheGluTyrGlnProAspProTrpAsnThrHisVal                          
705710715720                                                              
TGGAAAGGCACCAACGGGACCCCCACAAAGCGGCGAGCCATCGGCTTC2208                      
TrpLysGlyThrAsnGlyThrProThrLysArgArgAlaIleGlyPhe                          
725730735                                                                 
AAGAAGCTAGCAGAAGCTGTCAAGTTCTCGGCCAAGCTGATGGGGCAG2256                      
LysLysLeuAlaGluAlaValLysPheSerAlaLysLeuMetGlyGln                          
740745750                                                                 
GCTATGGCCAAGAGGGTGAAAGCGACCATCCTCTATGCCACAGAGACA2304                      
AlaMetAlaLysArgValLysAlaThrIleLeuTyrAlaThrGluThr                          
755760765                                                                 
GGCAAATCGCAAGCTTATGCCAAGACCTTGTGTGAGATCTTCAAACAC2352                      
GlyLysSerGlnAlaTyrAlaLysThrLeuCysGluIlePheLysHis                          
770775780                                                                 
GCCTTTGATGCCAAGGTGATGTCCATGGAAGAATATGACATTGTGCAC2400                      
AlaPheAspAlaLysValMetSerMetGluGluTyrAspIleValHis                          
785790795800                                                              
CTGGAACATGAAACTCTGGTCCTTGTGGTCACCAGCACCTTTGGCAAT2448                      
LeuGluHisGluThrLeuValLeuValValThrSerThrPheGlyAsn                          
805810815                                                                 
GGAGATCCCCCTGAGAATGGGGAGAAATTCGGCTGTGCTTTGATGGAA2496                      
GlyAspProProGluAsnGlyGluLysPheGlyCysAlaLeuMetGlu                          
820825830                                                                 
ATGAGGCACCCCAACTCTGTGCAGGAAGAAAGGAAGAGCTACAAGGTC2544                      
MetArgHisProAsnSerValGlnGluGluArgLysSerTyrLysVal                          
835840845                                                                 
CGATTCAACAGCGTCTCCTCCTACTCTGACTCCCAAAAATCATCAGGC2592                      
ArgPheAsnSerValSerSerTyrSerAspSerGlnLysSerSerGly                          
850855860                                                                 
GATGGGCCCGACCTCAGAGACAACTTTGAGAGTGCTGGACCCCTGGCC2640                      
AspGlyProAspLeuArgAspAsnPheGluSerAlaGlyProLeuAla                          
865870875880                                                              
AATGTGAGGTTCTCAGTTTTTGGCCTCGGCTCACGAGCATACCCTCAC2688                      
AsnValArgPheSerValPheGlyLeuGlySerArgAlaTyrProHis                          
885890895                                                                 
TTTTGCGCCTTCGGACACGCTGTGGACACCCTCCTGGAAGAACTGGGA2736                      
PheCysAlaPheGlyHisAlaValAspThrLeuLeuGluGluLeuGly                          
900905910                                                                 
GGGGAGAGGATCCTGAAGATGAGGGAAGGGGATGAGCTCTGTGGGCAG2784                      
GlyGluArgIleLeuLysMetArgGluGlyAspGluLeuCysGlyGln                          
915920925                                                                 
GAAGAGGCTTTCAGGACCTGGGCCAAGAAGGTCTTCAAGGCAGCCTGT2832                      
GluGluAlaPheArgThrTrpAlaLysLysValPheLysAlaAlaCys                          
930935940                                                                 
GATGTCTTCTGTGTGGGAGATGATGTCAACATTGAAAAGGCCAACAAT2880                      
AspValPheCysValGlyAspAspValAsnIleGluLysAlaAsnAsn                          
945950955960                                                              
TCCCTCATCAGCAATGATCGCAGCTGGAAGAGAAACAAGTTCCGCCTC2928                      
SerLeuIleSerAsnAspArgSerTrpLysArgAsnLysPheArgLeu                          
965970975                                                                 
ACCTTTGTGGCCGAAGCTCCAGAACTCACACAAGGTCTATCCAATGTC2976                      
ThrPheValAlaGluAlaProGluLeuThrGlnGlyLeuSerAsnVal                          
980985990                                                                 
CACAAAAAGCGAGTCTCAGCTGCCCGGCTCCTTAGCCGTCAAAACCTC3024                      
HisLysLysArgValSerAlaAlaArgLeuLeuSerArgGlnAsnLeu                          
99510001005                                                               
CAGAGCCCTAAATCCAGTCGGTCAACTATCTTCGTGCGTCTCCACACC3072                      
GlnSerProLysSerSerArgSerThrIlePheValArgLeuHisThr                          
101010151020                                                              
AACGGGAGCCAGGAGCTGCAGTACCAGCCTGGGGACCACCTGGGTGTC3120                      
AsnGlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeuGlyVal                          
1025103010351040                                                          
TTCCCTGGCAACCACGAGGACCTCGTGAATGCCCTGATCGAGCGGCTG3168                      
PheProGlyAsnHisGluAspLeuValAsnAlaLeuIleGluArgLeu                          
104510501055                                                              
GAGGACGCGCCGCCTGTCAACCAGATGGTGAAAGTGGAACTGCTGGAG3216                      
GluAspAlaProProValAsnGlnMetValLysValGluLeuLeuGlu                          
106010651070                                                              
GAGCGGAACACGGCTTTAGGTGTCATCAGTAACTGGACAGACGAGCTC3264                      
GluArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAspGluLeu                          
107510801085                                                              
CGCCTCCCACCCTGCACCATCTTCCAGGCCTTCAAGTACTACCTGGAC3312                      
ArgLeuProProCysThrIlePheGlnAlaPheLysTyrTyrLeuAsp                          
109010951100                                                              
ATCACCACGCCACCAACGCCCCTGCAGCTGCAGCAGTTTGCCTCCCTA3360                      
IleThrThrProProThrProLeuGlnLeuGlnGlnPheAlaSerLeu                          
1105111011151120                                                          
GCTACCAGCGAGAAGGAGAAGCAGCGTCTGCTGGTCCTCAGCAAGGGT3408                      
AlaThrSerGluLysGluLysGlnArgLeuLeuValLeuSerLysGly                          
112511301135                                                              
TTGCAGGAGTACGAGGAATGGAAATGGGGCAAGAACCCCACCATCGTG3456                      
LeuGlnGluTyrGluGluTrpLysTrpGlyLysAsnProThrIleVal                          
114011451150                                                              
GAGGTGCTGGAGGAGTTCCCATCTATCCAGATGCCGGCCACCCTGCTC3504                      
GluValLeuGluGluPheProSerIleGlnMetProAlaThrLeuLeu                          
115511601165                                                              
CTGACCCAGCTGTCCCTGCTGCAGCCCCGCTACTATTCCATCAGCTCC3552                      
LeuThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIleSerSer                          
117011751180                                                              
TCCCCAGACATGTACCCTGATGAAGTGCACCTCACTGTGGCCATCGTT3600                      
SerProAspMetTyrProAspGluValHisLeuThrValAlaIleVal                          
1185119011951200                                                          
TCCTACCGCACTCGAGATGGAGAAGGACCAATTCACCACGGCGTATGC3648                      
SerTyrArgThrArgAspGlyGluGlyProIleHisHisGlyValCys                          
120512101215                                                              
TCCTCCTGGCTCAACCGGATACAGGCTGACGAACTGGTCCCCTGTTTC3696                      
SerSerTrpLeuAsnArgIleGlnAlaAspGluLeuValProCysPhe                          
122012251230                                                              
GTGAGAGGAGCACCCAGCTTCCACCTGCCCCGGAACCCCCAAGTCCCC3744                      
ValArgGlyAlaProSerPheHisLeuProArgAsnProGlnValPro                          
123512401245                                                              
TGCATCCTCGTTGGACCAGGCACCGGCATTGCCCCTTTCCGAAGCTTC3792                      
CysIleLeuValGlyProGlyThrGlyIleAlaProPheArgSerPhe                          
125012551260                                                              
TGGCAACAGCGGCAATTTGATATCCAACACAAAGGAATGAACCCCTGC3840                      
TrpGlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsnProCys                          
1265127012751280                                                          
CCCATGGTCCTGGTCTTCGGGTGCCGGCAATCCAAGATAGATCATATC3888                      
ProMetValLeuValPheGlyCysArgGlnSerLysIleAspHisIle                          
128512901295                                                              
TACAGGGAAGAGACCCTGCAGGCCAAGAACAAGGGGGTCTTCAGAGAG3936                      
TyrArgGluGluThrLeuGlnAlaLysAsnLysGlyValPheArgGlu                          
130013051310                                                              
CTGTACACGGCTTACTCCCGGGAGCCAGACAAACCAAAGAAGTACGTG3984                      
LeuTyrThrAlaTyrSerArgGluProAspLysProLysLysTyrVal                          
131513201325                                                              
CAGGACATCCTGCAGGAGCAGCTGGCGGAGTCTGTGTACCGAGCCCTG4032                      
GlnAspIleLeuGlnGluGlnLeuAlaGluSerValTyrArgAlaLeu                          
133013351340                                                              
AAGGAGCAAGGGGGCCACATATACGTCTGTGGGGACGTCACCATGGCT4080                      
LysGluGlnGlyGlyHisIleTyrValCysGlyAspValThrMetAla                          
1345135013551360                                                          
GCTGATGTCCTCAAAGCCATCCAGCGCATCATGACCCAGCAGGGGAAG4128                      
AlaAspValLeuLysAlaIleGlnArgIleMetThrGlnGlnGlyLys                          
136513701375                                                              
CTCTCGGCAGAGGACGCCGGCGTATTCATCAGCCGGATGAGGGATGAC4176                      
LeuSerAlaGluAspAlaGlyValPheIleSerArgMetArgAspAsp                          
138013851390                                                              
AACCGATACCATGAGGATATTTTTGGAGTCACCCTGCGAACGTACGAA4224                      
AsnArgTyrHisGluAspIlePheGlyValThrLeuArgThrTyrGlu                          
139514001405                                                              
GTGACCAACCGCCTTAGATCTGAGTCCATTGCCTTCATTGAAGAGAGC4272                      
ValThrAsnArgLeuArgSerGluSerIleAlaPheIleGluGluSer                          
141014151420                                                              
AAAAAAGACACCGATGAGGTTTTCAGCTCCTAACTGGACCCTCTTGCCCA4322                    
LysLysAspThrAspGluValPheSerSer                                            
14251430143                                                               
GCCGGCTGCAAGTTTGTAAGCGCGGGACAGA4353                                       
(2) INFORMATION FOR SEQ ID NO:19:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1434 amino acids                                              
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                  
MetGluAspHisMetPheGlyValGlnGlnIleGlnProAsnValIle                          
151015                                                                    
SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal                          
202530                                                                    
LysGluArgValSerLysProProValIleIleSerAspLeuIleArg                          
354045                                                                    
GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle                          
505560                                                                    
LeuAlaValAsnGlyArgProLeuValAspLeuSerTyrAspSerAla                          
65707580                                                                  
LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle                          
859095                                                                    
LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr                          
100105110                                                                 
GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro                          
115120125                                                                 
ProThrLysAlaValAspLeuSerHisGlnProProAlaGlyLysGlu                          
130135140                                                                 
GlnProLeuAlaValAspGlyAlaSerGlyProGlyAsnGlyProGln                          
145150155160                                                              
HisAlaTyrAspAspGlyGlnGluAlaGlySerLeuProHisAlaAsn                          
165170175                                                                 
GlyLeuAlaProArgProProGlyGlnAspProAlaLysLysAlaThr                          
180185190                                                                 
ArgValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeuLysGlu                          
195200205                                                                 
IleGluProValLeuSerLeuLeuThrSerGlySerArgGlyValLys                          
210215220                                                                 
GlyGlyAlaProAlaLysAlaGluMetLysAspMetGlyIleGlnVal                          
225230235240                                                              
AspArgAspLeuAspGlyLysSerHisLysProLeuProLeuGlyVal                          
245250255                                                                 
GluAsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsnValPro                          
260265270                                                                 
ValValLeuAsnAsnProTyrSerGluLysGluGlnProProThrSer                          
275280285                                                                 
GlyLysGlnSerProThrLysAsnGlySerProSerLysCysProArg                          
290295300                                                                 
PheLeuLysValLysAsnTrpGluThrGluValValLeuThrAspThr                          
305310315320                                                              
LeuHisLeuLysSerThrLeuGluThrGlyCysThrGluTyrIleCys                          
325330335                                                                 
MetGlySerIleMetHisProSerGlnHisAlaArgArgProGluAsp                          
340345350                                                                 
ValArgThrLysGlyGlnLeuPheProLeuAlaLysGluPheIleAsp                          
355360365                                                                 
GlnTyrTyrSerSerIleLysArgPheGlySerLysAlaHisMetGlu                          
370375380                                                                 
ArgLeuGluGluValAsnLysGluIleAspThrThrSerThrTyrGln                          
385390395400                                                              
LeuLysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrpArgAsn                          
405410415                                                                 
AlaSerArgCysValGlyArgIleGlnTrpSerLysLeuGlnValPhe                          
420425430                                                                 
AspAlaArgAspCysThrThrAlaHisGlyMetPheAsnTyrIleCys                          
435440445                                                                 
AsnHisValLysTyrAlaThrAsnLysGlyAsnLeuArgSerAlaIle                          
450455460                                                                 
ThrIlePheProGlnArgThrAspGlyLysHisAspPheArgValTrp                          
465470475480                                                              
AsnSerGlnLeuIleArgTyrAlaGlyTyrLysGlnProAspGlySer                          
485490495                                                                 
ThrLeuGlyAspProAlaAsnValGlnPheThrGluIleCysIleGln                          
500505510                                                                 
GlnGlyTrpLysProProArgGlyArgPheAspValLeuProLeuLeu                          
515520525                                                                 
LeuGlnAlaAsnGlyAsnAspProGluLeuPheGlnIleProProGlu                          
530535540                                                                 
LeuValLeuGluValProIleArgHisProLysPheGluTrpPheLys                          
545550555560                                                              
AspLeuGlyLeuLysTrpTyrGlyLeuProAlaValSerAsnMetLeu                          
565570575                                                                 
LeuGluIleGlyGlyLeuGluPheSerAlaCysProPheSerGlyTrp                          
580585590                                                                 
TyrMetGlyThrGluIleGlyValArgAspTyrCysAspAsnSerArg                          
595600605                                                                 
TyrAsnIleLeuGluGluValAlaLysLysMetAsnLeuAspMetArg                          
610615620                                                                 
LysThrSerSerLeuTrpLysAspGlnAlaLeuValGluIleAsnIle                          
625630635640                                                              
AlaValLeuTyrSerPheGlnSerAspLysValThrIleValAspHis                          
645650655                                                                 
HisSerAlaThrGluSerPheIleLysHisMetGluAsnGluTyrArg                          
660665670                                                                 
CysArgGlyGlyCysProAlaAspTrpValTrpIleValProProMet                          
675680685                                                                 
SerGlySerIleThrProValPheHisGlnGluMetLeuAsnTyrArg                          
690695700                                                                 
LeuThrProSerPheGluTyrGlnProAspProTrpAsnThrHisVal                          
705710715720                                                              
TrpLysGlyThrAsnGlyThrProThrLysArgArgAlaIleGlyPhe                          
725730735                                                                 
LysLysLeuAlaGluAlaValLysPheSerAlaLysLeuMetGlyGln                          
740745750                                                                 
AlaMetAlaLysArgValLysAlaThrIleLeuTyrAlaThrGluThr                          
755760765                                                                 
GlyLysSerGlnAlaTyrAlaLysThrLeuCysGluIlePheLysHis                          
770775780                                                                 
AlaPheAspAlaLysValMetSerMetGluGluTyrAspIleValHis                          
785790795800                                                              
LeuGluHisGluThrLeuValLeuValValThrSerThrPheGlyAsn                          
805810815                                                                 
GlyAspProProGluAsnGlyGluLysPheGlyCysAlaLeuMetGlu                          
820825830                                                                 
MetArgHisProAsnSerValGlnGluGluArgLysSerTyrLysVal                          
835840845                                                                 
ArgPheAsnSerValSerSerTyrSerAspSerGlnLysSerSerGly                          
850855860                                                                 
AspGlyProAspLeuArgAspAsnPheGluSerAlaGlyProLeuAla                          
865870875880                                                              
AsnValArgPheSerValPheGlyLeuGlySerArgAlaTyrProHis                          
885890895                                                                 
PheCysAlaPheGlyHisAlaValAspThrLeuLeuGluGluLeuGly                          
900905910                                                                 
GlyGluArgIleLeuLysMetArgGluGlyAspGluLeuCysGlyGln                          
915920925                                                                 
GluGluAlaPheArgThrTrpAlaLysLysValPheLysAlaAlaCys                          
930935940                                                                 
AspValPheCysValGlyAspAspValAsnIleGluLysAlaAsnAsn                          
945950955960                                                              
SerLeuIleSerAsnAspArgSerTrpLysArgAsnLysPheArgLeu                          
965970975                                                                 
ThrPheValAlaGluAlaProGluLeuThrGlnGlyLeuSerAsnVal                          
980985990                                                                 
HisLysLysArgValSerAlaAlaArgLeuLeuSerArgGlnAsnLeu                          
99510001005                                                               
GlnSerProLysSerSerArgSerThrIlePheValArgLeuHisThr                          
101010151020                                                              
AsnGlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeuGlyVal                          
1025103010351040                                                          
PheProGlyAsnHisGluAspLeuValAsnAlaLeuIleGluArgLeu                          
104510501055                                                              
GluAspAlaProProValAsnGlnMetValLysValGluLeuLeuGlu                          
106010651070                                                              
GluArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAspGluLeu                          
107510801085                                                              
ArgLeuProProCysThrIlePheGlnAlaPheLysTyrTyrLeuAsp                          
109010951100                                                              
IleThrThrProProThrProLeuGlnLeuGlnGlnPheAlaSerLeu                          
1105111011151120                                                          
AlaThrSerGluLysGluLysGlnArgLeuLeuValLeuSerLysGly                          
112511301135                                                              
LeuGlnGluTyrGluGluTrpLysTrpGlyLysAsnProThrIleVal                          
114011451150                                                              
GluValLeuGluGluPheProSerIleGlnMetProAlaThrLeuLeu                          
115511601165                                                              
LeuThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIleSerSer                          
117011751180                                                              
SerProAspMetTyrProAspGluValHisLeuThrValAlaIleVal                          
1185119011951200                                                          
SerTyrArgThrArgAspGlyGluGlyProIleHisHisGlyValCys                          
120512101215                                                              
SerSerTrpLeuAsnArgIleGlnAlaAspGluLeuValProCysPhe                          
122012251230                                                              
ValArgGlyAlaProSerPheHisLeuProArgAsnProGlnValPro                          
123512401245                                                              
CysIleLeuValGlyProGlyThrGlyIleAlaProPheArgSerPhe                          
125012551260                                                              
TrpGlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsnProCys                          
1265127012751280                                                          
ProMetValLeuValPheGlyCysArgGlnSerLysIleAspHisIle                          
128512901295                                                              
TyrArgGluGluThrLeuGlnAlaLysAsnLysGlyValPheArgGlu                          
130013051310                                                              
LeuTyrThrAlaTyrSerArgGluProAspLysProLysLysTyrVal                          
131513201325                                                              
GlnAspIleLeuGlnGluGlnLeuAlaGluSerValTyrArgAlaLeu                          
133013351340                                                              
LysGluGlnGlyGlyHisIleTyrValCysGlyAspValThrMetAla                          
1345135013551360                                                          
AlaAspValLeuLysAlaIleGlnArgIleMetThrGlnGlnGlyLys                          
136513701375                                                              
LeuSerAlaGluAspAlaGlyValPheIleSerArgMetArgAspAsp                          
138013851390                                                              
AsnArgTyrHisGluAspIlePheGlyValThrLeuArgThrTyrGlu                          
139514001405                                                              
ValThrAsnArgLeuArgSerGluSerIleAlaPheIleGluGluSer                          
141014151420                                                              
LysLysAspThrAspGluValPheSerSer                                            
14251430                                                                  
(2) INFORMATION FOR SEQ ID NO:20:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 4780 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: Human NOS-SN gene, Nakane, et al,                 
FEBS Lett 316:175 (1993)                                                  
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 431..4732                                                   
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                  
GAGCGGACGGGCTCATGATGCCTCAGATCTGATCCGCATCTAACAGGCTGGCAATGAAGA60            
TACCCAGAGAATAGTTCACATCTATCATGCGTCACTTCTAGACACAGCCATCAGACGCAT120           
CTCCTCCCCTTTCTGCCTGACCTTAGGACACGTCCCACCGCCTCTCTTGACGTCTGCCTG180           
GTCAACCATCACTTCCTTAGAGAATAAGGAGAGAGGCGGATGCAGGAAATCATGCCACCG240           
ACGGGCCACCAGCCATGAGTGGGTGACGCTGAGCTGACGTCAAAGACAGAGAGGGCTGAA300           
GCCTTGTCAGCACCTGTCACCCCGGCTCCTGCTCTCCGTGTAGCCTGAAGCCTGGATCCT360           
CCTGGTGAAATCATCTTGGCCTGATAGCATTGTGAGGTCTTCAGACAGGACCCCTCGGAA420           
GCTAGTTACCATGGAGGATCACATGTTCGGTGTTCAGCAAATCCAGCCC469                      
MetGluAspHisMetPheGlyValGlnGlnIleGlnPro                                   
1510                                                                      
AATGTCATTTCTGTTCGTCTCTTCAAGCGCAAAGTTGGGGGCCTGGGA517                       
AsnValIleSerValArgLeuPheLysArgLysValGlyGlyLeuGly                          
152025                                                                    
TTTCTGGTGAAGGAGCGGGTCAGTAAGCCGCCCGTGATCATCTCTGAC565                       
PheLeuValLysGluArgValSerLysProProValIleIleSerAsp                          
30354045                                                                  
CTGATTCGTGGGGGCGCCGCAGAGCAGAGTGGCCTCATCCAGGCCGGA613                       
LeuIleArgGlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGly                          
505560                                                                    
GACATCATTCTTGCGGTCAACGGCCGGCCCTTGGTGGACCTGAGCTAT661                       
AspIleIleLeuAlaValAsnGlyArgProLeuValAspLeuSerTyr                          
657075                                                                    
GACAGCGCCCTGGAGGTACTCAGAGGCATTGCCTCTGAGACCCACGTG709                       
AspSerAlaLeuGluValLeuArgGlyIleAlaSerGluThrHisVal                          
808590                                                                    
GTCCTCATTCTGAGGGGCCCTGAAGGTTTCACCACGCACCTGGAGACC757                       
ValLeuIleLeuArgGlyProGluGlyPheThrThrHisLeuGluThr                          
95100105                                                                  
ACCTTTACAGGTGATGGGACCCCCAAGACCATCCGGGTGACACAGCCC805                       
ThrPheThrGlyAspGlyThrProLysThrIleArgValThrGlnPro                          
110115120125                                                              
CTGGGTCCCCCCACCAAAGCCGTGGATCTGTCCCACCAGCCACCGGCC853                       
LeuGlyProProThrLysAlaValAspLeuSerHisGlnProProAla                          
130135140                                                                 
GGCAAAGAACAGCCCCTGGCAGTGGATGGGGCCTCGGGTCCCGGGAAT901                       
GlyLysGluGlnProLeuAlaValAspGlyAlaSerGlyProGlyAsn                          
145150155                                                                 
GGGCCTCAGCATGCCTACGATGATGGGCAGGAGGCTGGCTCACTCCCC949                       
GlyProGlnHisAlaTyrAspAspGlyGlnGluAlaGlySerLeuPro                          
160165170                                                                 
CATGCCAACGGCTGGCCCCAGGCCCCCAGGCAGGACCCCGCGAAGAAA997                       
HisAlaAsnGlyTrpProGlnAlaProArgGlnAspProAlaLysLys                          
175180185                                                                 
GCAACCAGAGTCAGCCTCCAAGGCAGAGGGGAGAACAATGAACTGCTC1045                      
AlaThrArgValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeu                          
190195200205                                                              
AAGGAGATAGAGCCTGTGCTGAGCCTTCTCACCAGTGGGAGCAGAGGG1093                      
LysGluIleGluProValLeuSerLeuLeuThrSerGlySerArgGly                          
210215220                                                                 
GTCAAGGGAGGGGCACCTGCCAAGGCAGAGATGAAAGATATGGGAATC1141                      
ValLysGlyGlyAlaProAlaLysAlaGluMetLysAspMetGlyIle                          
225230235                                                                 
CAGGTGGACAGAGATTTGGACGGCAAGTCACACAAACCTCTGCCCCTC1189                      
GlnValAspArgAspLeuAspGlyLysSerHisLysProLeuProLeu                          
240245250                                                                 
GGCGTGGAGAACGACCGAGTCTTCAATGACCTATGGGGGAAGGGCAAT1237                      
GlyValGluAsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsn                          
255260265                                                                 
GTGCCTGTCGTCCTCAACAACCCATATTCAGAGAAGGAGCAGCCCCCC1285                      
ValProValValLeuAsnAsnProTyrSerGluLysGluGlnProPro                          
270275280285                                                              
ACCTCAGGAAAACAGTCCCCCACAAAGAATGGCAGCCCCTCCAAGTGT1333                      
ThrSerGlyLysGlnSerProThrLysAsnGlySerProSerLysCys                          
290295300                                                                 
CCACGCTTCCTCAAGGTCAAGAACTGGGAGACTGAGGTGGTTCTCACT1381                      
ProArgPheLeuLysValLysAsnTrpGluThrGluValValLeuThr                          
305310315                                                                 
GACACCCTCCACCTTAAGAGCACATTGGAAACGGGATGCACTGAGTAC1429                      
AspThrLeuHisLeuLysSerThrLeuGluThrGlyCysThrGluTyr                          
320325330                                                                 
ATCTGCATGGGCTCCATCATGCATCCTTCTCAGCATGCAAGGAGGCCT1477                      
IleCysMetGlySerIleMetHisProSerGlnHisAlaArgArgPro                          
335340345                                                                 
GAAGACGTCCGCACAAAAGGACAGCTCTTCCCTCTCGCCAAAGAGTTT1525                      
GluAspValArgThrLysGlyGlnLeuPheProLeuAlaLysGluPhe                          
350355360365                                                              
ATTGATCAATACTATTCATCAATTAAAAGATTTGGCTCCAAAGCCCAC1573                      
IleAspGlnTyrTyrSerSerIleLysArgPheGlySerLysAlaHis                          
370375380                                                                 
ATGGAAAGGCTGGAAGAGGTGAACAAAGAGATCGACACCACTAGCACT1621                      
MetGluArgLeuGluGluValAsnLysGluIleAspThrThrSerThr                          
385390395                                                                 
TACCAGCTCAAGGACACAGAGCTCATCTATGGGGCCAAGCACGCCTGG1669                      
TyrGlnLeuLysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrp                          
400405410                                                                 
CGGAATGCCTCGCGCTGTGTGGGCAGGATCCAGTGGTCCAAGCTGCAG1717                      
ArgAsnAlaSerArgCysValGlyArgIleGlnTrpSerLysLeuGln                          
415420425                                                                 
GTATTCGATGCCCGTGACTGCACCACGGCCCACGGGATGTTCAACTAC1765                      
ValPheAspAlaArgAspCysThrThrAlaHisGlyMetPheAsnTyr                          
430435440445                                                              
ATCTGTAACCATGTCAAGTATGCCACCAACAAAGGGAACCTCAGGTCT1813                      
IleCysAsnHisValLysTyrAlaThrAsnLysGlyAsnLeuArgSer                          
450455460                                                                 
GCCATCACCATATTCCCCCAGAGGACAGACGGCAAGCACGACTTCCGA1861                      
AlaIleThrIlePheProGlnArgThrAspGlyLysHisAspPheArg                          
465470475                                                                 
GTCTGGAACTCCCAGCTCATCCGCTACGCTGGCTACAAGCACCGTGAC1909                      
ValTrpAsnSerGlnLeuIleArgTyrAlaGlyTyrLysHisArgAsp                          
480485490                                                                 
GGCTCCACCCTGGGGGACCCAGCCAATGTGCAGTTCACAGAGATATGC1957                      
GlySerThrLeuGlyAspProAlaAsnValGlnPheThrGluIleCys                          
495500505                                                                 
ATACAGCAGGGCTGGAAACCGCCTAGAGGCCGCTTCGATGTCCTGCCG2005                      
IleGlnGlnGlyTrpLysProProArgGlyArgPheAspValLeuPro                          
510515520525                                                              
CTCCTGCTTCAGGCCAACGGCAATGACCCTGAGCTCTTCCAGATTCCT2053                      
LeuLeuLeuGlnAlaAsnGlyAsnAspProGluLeuPheGlnIlePro                          
530535540                                                                 
CCAGAGCTGGTGTTGGAACTTCCCATCAGGCACCCCAAGTTTGAGTGG2101                      
ProGluLeuValLeuGluLeuProIleArgHisProLysPheGluTrp                          
545550555                                                                 
TTCAAGGACCTGGCGCTGAAGTGGTACGGCCTCCCCGCCGTGTCCAAC2149                      
PheLysAspLeuAlaLeuLysTrpTyrGlyLeuProAlaValSerAsn                          
560565570                                                                 
ATGCTCCTAGAGATTGGCGGCCTGGAGTTCAGCGCCTGTCCCTTCAGT2197                      
MetLeuLeuGluIleGlyGlyLeuGluPheSerAlaCysProPheSer                          
575580585                                                                 
GGCTGGTACATGGGCACAGAGATTGGTGTCCGCGACTACTGTGACAAC2245                      
GlyTrpTyrMetGlyThrGluIleGlyValArgAspTyrCysAspAsn                          
590595600605                                                              
TCCCGCTACAATATCCTGGAGGAAGTGGCCAAGAAGATGAACTTAGAC2293                      
SerArgTyrAsnIleLeuGluGluValAlaLysLysMetAsnLeuAsp                          
610615620                                                                 
ATGAGGAAGACGTCCTCCCTGTGGAAGGACCAGGCGCTGGTGGAGATC2341                      
MetArgLysThrSerSerLeuTrpLysAspGlnAlaLeuValGluIle                          
625630635                                                                 
AATATCGCGGTTCTCTATAGCTTCCAGAGTGACAAAGTGACCATTGTT2389                      
AsnIleAlaValLeuTyrSerPheGlnSerAspLysValThrIleVal                          
640645650                                                                 
GACCATCACTCCGCCACCGAGTCCTTCATTAAGCACATGGAGAATGAG2437                      
AspHisHisSerAlaThrGluSerPheIleLysHisMetGluAsnGlu                          
655660665                                                                 
TACCGCTGCCGGGGGGGCTGCCCTGCCGACTGGGTGTGGATCGTGCCC2485                      
TyrArgCysArgGlyGlyCysProAlaAspTrpValTrpIleValPro                          
670675680685                                                              
CCCATGTCCGGAAGCATCACCCCTGTGTTCCACCAGGAGATGCTCAAC2533                      
ProMetSerGlySerIleThrProValPheHisGlnGluMetLeuAsn                          
690695700                                                                 
TACCGGCTCACCCCCTCCTTCGAATACCAGCCTGATCCCTGGAACACG2581                      
TyrArgLeuThrProSerPheGluTyrGlnProAspProTrpAsnThr                          
705710715                                                                 
CATGTCTGGAAAGGCACCAACGGGACCCCCACAAAGCGGCGAGCCATC2629                      
HisValTrpLysGlyThrAsnGlyThrProThrLysArgArgAlaIle                          
720725730                                                                 
GGCTTCAAGAAGCTAGCAGAAGCTGTCAAGTTCTCGGCCAAGCTGATG2677                      
GlyPheLysLysLeuAlaGluAlaValLysPheSerAlaLysLeuMet                          
735740745                                                                 
GGGCAGGCTATGGCCAAGAGGGTGAAAGCGACCATCCTCTATGCCACA2725                      
GlyGlnAlaMetAlaLysArgValLysAlaThrIleLeuTyrAlaThr                          
750755760765                                                              
GAGACAGGCAAATCGCAAGCTTATGCCAAGACCTTGTGTGAGATCTTC2773                      
GluThrGlyLysSerGlnAlaTyrAlaLysThrLeuCysGluIlePhe                          
770775780                                                                 
AAACACGCCTTTGATGCCAAGGTGATGTCCATGGAAGAATATGACATT2821                      
LysHisAlaPheAspAlaLysValMetSerMetGluGluTyrAspIle                          
785790795                                                                 
GTGCACCTGGAACATGAAACTCTGGTCCTTGTGGTCACCAGCACCTTT2869                      
ValHisLeuGluHisGluThrLeuValLeuValValThrSerThrPhe                          
800805810                                                                 
GGCAATGGAGATCCCCCTGAGAATGGGGAGAAATTCGGCTGTGCTTTG2917                      
GlyAsnGlyAspProProGluAsnGlyGluLysPheGlyCysAlaLeu                          
815820825                                                                 
ATGGAAATGAGGCACCCCAACTCTGTGCAGGAAGAAAGGAAGAGCTAC2965                      
MetGluMetArgHisProAsnSerValGlnGluGluArgLysSerTyr                          
830835840845                                                              
AAGGTCCGATTCAACAGCGTCTCCTCCTACTCTGACTCCCAAAAATCA3013                      
LysValArgPheAsnSerValSerSerTyrSerAspSerGlnLysSer                          
850855860                                                                 
TCAGGCGATGGGCCCGACCTCAGAGACAACTTTGAGAGTGCTGGACCC3061                      
SerGlyAspGlyProAspLeuArgAspAsnPheGluSerAlaGlyPro                          
865870875                                                                 
CTGGCCAATGTGAGGTTCTCAGTTTTTGGCCTCGGCTCACGAGCATAC3109                      
LeuAlaAsnValArgPheSerValPheGlyLeuGlySerArgAlaTyr                          
880885890                                                                 
CCTCACTTTTGCGCCTTCGGACACGCTGTGGACACCCTCCTGGAAGAA3157                      
ProHisPheCysAlaPheGlyHisAlaValAspThrLeuLeuGluGlu                          
895900905                                                                 
CTGGGAGGGGAGAGGATCCTGAAGATGAGGGAAGGGGATGAGCTCTGT3205                      
LeuGlyGlyGluArgIleLeuLysMetArgGluGlyAspGluLeuCys                          
910915920925                                                              
GGGCAGGAAGAGGCTTTCAGGACCTGGGCCAAGAAGGTCTTCAAGGCA3253                      
GlyGlnGluGluAlaPheArgThrTrpAlaLysLysValPheLysAla                          
930935940                                                                 
GCCTGTGATGTCTTCTGTGTGGGAGATGATGTCAACATTGAAAAGGCC3301                      
AlaCysAspValPheCysValGlyAspAspValAsnIleGluLysAla                          
945950955                                                                 
AACAATTCCCTCATCAGCAATGATCGCAGCTGGAAGAGAAACAAGTTC3349                      
AsnAsnSerLeuIleSerAsnAspArgSerTrpLysArgAsnLysPhe                          
960965970                                                                 
CGCCTCACCTTTGTGGCCGAAGCTCCAGAACTCACACAAGGTCTATCC3397                      
ArgLeuThrPheValAlaGluAlaProGluLeuThrGlnGlyLeuSer                          
975980985                                                                 
AATGTCCACAAAAAGCGAGTCTCAGCTGCCCGGCTCCTTAGCCGTCAA3445                      
AsnValHisLysLysArgValSerAlaAlaArgLeuLeuSerArgGln                          
99099510001005                                                            
AACCTCCAGAGCCCTAAATCCAGTCGGTCAACTATCTTCGTGCGTCTC3493                      
AsnLeuGlnSerProLysSerSerArgSerThrIlePheValArgLeu                          
101010151020                                                              
CACACCAACGGGAGCCAGGAGCTGCAGTACCAGCCTGGGGACCACCTG3541                      
HisThrAsnGlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeu                          
102510301035                                                              
GGTGTCTTCCCTGGCAACCACGAGGACCTCGTGAATGCCCTGATCGAG3589                      
GlyValPheProGlyAsnHisGluAspLeuValAsnAlaLeuIleGlu                          
104010451050                                                              
CGGCTGGAGGACGCGCCGCCTGTCAACCAGATGGTGAAAGTGGAACTG3637                      
ArgLeuGluAspAlaProProValAsnGlnMetValLysValGluLeu                          
105510601065                                                              
CTGGAGGAGCGGAACACGGCTTTAGGTGTCATCAGTAACTGGACAGAC3685                      
LeuGluGluArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAsp                          
1070107510801085                                                          
GAGCTCCGCCTCCCGCCCTGCACCATCTTCCAGGCCTTCAAGTACTAC3733                      
GluLeuArgLeuProProCysThrIlePheGlnAlaPheLysTyrTyr                          
109010951100                                                              
CTGGACATCACCACGCCACCAACGCCTCTGCAGCTGCAGCAGTTTGCC3781                      
LeuAspIleThrThrProProThrProLeuGlnLeuGlnGlnPheAla                          
110511101115                                                              
TCCCTAGCTACCAGCGAGAAGGAGAAGCAGCGTCTGCTGGTCCTCAGC3829                      
SerLeuAlaThrSerGluLysGluLysGlnArgLeuLeuValLeuSer                          
112011251130                                                              
AAGGGTTTGCAGGAGTACGAGGAATGGAAATGGGGCAAGAACCCCACC3877                      
LysGlyLeuGlnGluTyrGluGluTrpLysTrpGlyLysAsnProThr                          
113511401145                                                              
ATCGTGGAGGTGCTGGAGGAGTTCCCATCTATCCAGATGCCGGCCACC3925                      
IleValGluValLeuGluGluPheProSerIleGlnMetProAlaThr                          
1150115511601165                                                          
CTGCTCCTGACCCAGCTGTCCCTGCTGCAGCCCCGCTACTATTCCATC3973                      
LeuLeuLeuThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIle                          
117011751180                                                              
AGCTCCTCCCCAGACATGTACCCTGATGAAGTGCACCTCACTGTGGCC4021                      
SerSerSerProAspMetTyrProAspGluValHisLeuThrValAla                          
118511901195                                                              
ATCGTTTCCTACCGCACTCGAGATGGAGAAGGACCAATTCACCACGGC4069                      
IleValSerTyrArgThrArgAspGlyGluGlyProIleHisHisGly                          
120012051210                                                              
GTATGCTCCTCCTGGCTCAACCGGATACAGGCTGACGAACTGGTCCCC4117                      
ValCysSerSerTrpLeuAsnArgIleGlnAlaAspGluLeuValPro                          
121512201225                                                              
TGTTTCGTGAGAGGAGCACCCAGCTTCCACCTGCCCCGGAACCCCCAA4165                      
CysPheValArgGlyAlaProSerPheHisLeuProArgAsnProGln                          
1230123512401245                                                          
GTCCCCTGCATCCTCGTTGGACCAGGCACCGGCATTGCCCCTTTCCGA4213                      
ValProCysIleLeuValGlyProGlyThrGlyIleAlaProPheArg                          
125012551260                                                              
AGCTTCTGGCAACAGCGGCAATTTGATATCCAACACAAAGGAATGAAC4261                      
SerPheTrpGlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsn                          
126512701275                                                              
CCCTGCCCCATGGTCCTGGTCTTCGGGTGCCGGCAATCCAAGATAGAT4309                      
ProCysProMetValLeuValPheGlyCysArgGlnSerLysIleAsp                          
128012851290                                                              
CATATCTACAGGGAAGAGACCCTGCAGGCCAAGAACAAGGGGGTCTTC4357                      
HisIleTyrArgGluGluThrLeuGlnAlaLysAsnLysGlyValPhe                          
129513001305                                                              
AGAGAGCTGTACACGGCTTACTCCCGGGAGCCAGACAAACCAAAGAAG4405                      
ArgGluLeuTyrThrAlaTyrSerArgGluProAspLysProLysLys                          
1310131513201325                                                          
TACGTGCAGGACATCCTGCAGGAGCAGCTGGCGGAGTCTGTGTACCGA4453                      
TyrValGlnAspIleLeuGlnGluGlnLeuAlaGluSerValTyrArg                          
133013351340                                                              
GCCCTGAAGGAGCAAGGGGGCCACATATACGTCTGTGGGGACGTCACC4501                      
AlaLeuLysGluGlnGlyGlyHisIleTyrValCysGlyAspValThr                          
134513501355                                                              
ATGGCTGCTGATGTCCTCAAAGCCATCCAGCGCATCATGACCCAGCAG4549                      
MetAlaAlaAspValLeuLysAlaIleGlnArgIleMetThrGlnGln                          
136013651370                                                              
GGGAAGCTCTCGGCAGAGGACGCCGGCGTATTCATCAGCCGGATGAGG4597                      
GlyLysLeuSerAlaGluAspAlaGlyValPheIleSerArgMetArg                          
137513801385                                                              
GATGACAACCGATACCATGAGGATATTTTTGGAGTCACCCTGCGAACG4645                      
AspAspAsnArgTyrHisGluAspIlePheGlyValThrLeuArgThr                          
1390139514001405                                                          
ATCGAAGTGACCAACCGCCTTAGATCTGAGTCCATTGCCTTCATTGAA4693                      
IleGluValThrAsnArgLeuArgSerGluSerIleAlaPheIleGlu                          
141014151420                                                              
GAGAGCAAAAAAGACACCGATGAGGTTTTCAGCTCCTAACTGGACC4739                        
GluSerLysLysAspThrAspGluValPheSerSer                                      
14251430                                                                  
CTCTTGCCCAGCCGGCTGCAAGTTTGTAAGCGCGGGACAGA4780                             
(2) INFORMATION FOR SEQ ID NO:21:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1433 amino acids                                              
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                  
MetGluAspHisMetPheGlyValGlnGlnIleGlnProAsnValIle                          
151015                                                                    
SerValArgLeuPheLysArgLysValGlyGlyLeuGlyPheLeuVal                          
202530                                                                    
LysGluArgValSerLysProProValIleIleSerAspLeuIleArg                          
354045                                                                    
GlyGlyAlaAlaGluGlnSerGlyLeuIleGlnAlaGlyAspIleIle                          
505560                                                                    
LeuAlaValAsnGlyArgProLeuValAspLeuSerTyrAspSerAla                          
65707580                                                                  
LeuGluValLeuArgGlyIleAlaSerGluThrHisValValLeuIle                          
859095                                                                    
LeuArgGlyProGluGlyPheThrThrHisLeuGluThrThrPheThr                          
100105110                                                                 
GlyAspGlyThrProLysThrIleArgValThrGlnProLeuGlyPro                          
115120125                                                                 
ProThrLysAlaValAspLeuSerHisGlnProProAlaGlyLysGlu                          
130135140                                                                 
GlnProLeuAlaValAspGlyAlaSerGlyProGlyAsnGlyProGln                          
145150155160                                                              
HisAlaTyrAspAspGlyGlnGluAlaGlySerLeuProHisAlaAsn                          
165170175                                                                 
GlyTrpProGlnAlaProArgGlnAspProAlaLysLysAlaThrArg                          
180185190                                                                 
ValSerLeuGlnGlyArgGlyGluAsnAsnGluLeuLeuLysGluIle                          
195200205                                                                 
GluProValLeuSerLeuLeuThrSerGlySerArgGlyValLysGly                          
210215220                                                                 
GlyAlaProAlaLysAlaGluMetLysAspMetGlyIleGlnValAsp                          
225230235240                                                              
ArgAspLeuAspGlyLysSerHisLysProLeuProLeuGlyValGlu                          
245250255                                                                 
AsnAspArgValPheAsnAspLeuTrpGlyLysGlyAsnValProVal                          
260265270                                                                 
ValLeuAsnAsnProTyrSerGluLysGluGlnProProThrSerGly                          
275280285                                                                 
LysGlnSerProThrLysAsnGlySerProSerLysCysProArgPhe                          
290295300                                                                 
LeuLysValLysAsnTrpGluThrGluValValLeuThrAspThrLeu                          
305310315320                                                              
HisLeuLysSerThrLeuGluThrGlyCysThrGluTyrIleCysMet                          
325330335                                                                 
GlySerIleMetHisProSerGlnHisAlaArgArgProGluAspVal                          
340345350                                                                 
ArgThrLysGlyGlnLeuPheProLeuAlaLysGluPheIleAspGln                          
355360365                                                                 
TyrTyrSerSerIleLysArgPheGlySerLysAlaHisMetGluArg                          
370375380                                                                 
LeuGluGluValAsnLysGluIleAspThrThrSerThrTyrGlnLeu                          
385390395400                                                              
LysAspThrGluLeuIleTyrGlyAlaLysHisAlaTrpArgAsnAla                          
405410415                                                                 
SerArgCysValGlyArgIleGlnTrpSerLysLeuGlnValPheAsp                          
420425430                                                                 
AlaArgAspCysThrThrAlaHisGlyMetPheAsnTyrIleCysAsn                          
435440445                                                                 
HisValLysTyrAlaThrAsnLysGlyAsnLeuArgSerAlaIleThr                          
450455460                                                                 
IlePheProGlnArgThrAspGlyLysHisAspPheArgValTrpAsn                          
465470475480                                                              
SerGlnLeuIleArgTyrAlaGlyTyrLysHisArgAspGlySerThr                          
485490495                                                                 
LeuGlyAspProAlaAsnValGlnPheThrGluIleCysIleGlnGln                          
500505510                                                                 
GlyTrpLysProProArgGlyArgPheAspValLeuProLeuLeuLeu                          
515520525                                                                 
GlnAlaAsnGlyAsnAspProGluLeuPheGlnIleProProGluLeu                          
530535540                                                                 
ValLeuGluLeuProIleArgHisProLysPheGluTrpPheLysAsp                          
545550555560                                                              
LeuAlaLeuLysTrpTyrGlyLeuProAlaValSerAsnMetLeuLeu                          
565570575                                                                 
GluIleGlyGlyLeuGluPheSerAlaCysProPheSerGlyTrpTyr                          
580585590                                                                 
MetGlyThrGluIleGlyValArgAspTyrCysAspAsnSerArgTyr                          
595600605                                                                 
AsnIleLeuGluGluValAlaLysLysMetAsnLeuAspMetArgLys                          
610615620                                                                 
ThrSerSerLeuTrpLysAspGlnAlaLeuValGluIleAsnIleAla                          
625630635640                                                              
ValLeuTyrSerPheGlnSerAspLysValThrIleValAspHisHis                          
645650655                                                                 
SerAlaThrGluSerPheIleLysHisMetGluAsnGluTyrArgCys                          
660665670                                                                 
ArgGlyGlyCysProAlaAspTrpValTrpIleValProProMetSer                          
675680685                                                                 
GlySerIleThrProValPheHisGlnGluMetLeuAsnTyrArgLeu                          
690695700                                                                 
ThrProSerPheGluTyrGlnProAspProTrpAsnThrHisValTrp                          
705710715720                                                              
LysGlyThrAsnGlyThrProThrLysArgArgAlaIleGlyPheLys                          
725730735                                                                 
LysLeuAlaGluAlaValLysPheSerAlaLysLeuMetGlyGlnAla                          
740745750                                                                 
MetAlaLysArgValLysAlaThrIleLeuTyrAlaThrGluThrGly                          
755760765                                                                 
LysSerGlnAlaTyrAlaLysThrLeuCysGluIlePheLysHisAla                          
770775780                                                                 
PheAspAlaLysValMetSerMetGluGluTyrAspIleValHisLeu                          
785790795800                                                              
GluHisGluThrLeuValLeuValValThrSerThrPheGlyAsnGly                          
805810815                                                                 
AspProProGluAsnGlyGluLysPheGlyCysAlaLeuMetGluMet                          
820825830                                                                 
ArgHisProAsnSerValGlnGluGluArgLysSerTyrLysValArg                          
835840845                                                                 
PheAsnSerValSerSerTyrSerAspSerGlnLysSerSerGlyAsp                          
850855860                                                                 
GlyProAspLeuArgAspAsnPheGluSerAlaGlyProLeuAlaAsn                          
865870875880                                                              
ValArgPheSerValPheGlyLeuGlySerArgAlaTyrProHisPhe                          
885890895                                                                 
CysAlaPheGlyHisAlaValAspThrLeuLeuGluGluLeuGlyGly                          
900905910                                                                 
GluArgIleLeuLysMetArgGluGlyAspGluLeuCysGlyGlnGlu                          
915920925                                                                 
GluAlaPheArgThrTrpAlaLysLysValPheLysAlaAlaCysAsp                          
930935940                                                                 
ValPheCysValGlyAspAspValAsnIleGluLysAlaAsnAsnSer                          
945950955960                                                              
LeuIleSerAsnAspArgSerTrpLysArgAsnLysPheArgLeuThr                          
965970975                                                                 
PheValAlaGluAlaProGluLeuThrGlnGlyLeuSerAsnValHis                          
980985990                                                                 
LysLysArgValSerAlaAlaArgLeuLeuSerArgGlnAsnLeuGln                          
99510001005                                                               
SerProLysSerSerArgSerThrIlePheValArgLeuHisThrAsn                          
101010151020                                                              
GlySerGlnGluLeuGlnTyrGlnProGlyAspHisLeuGlyValPhe                          
1025103010351040                                                          
ProGlyAsnHisGluAspLeuValAsnAlaLeuIleGluArgLeuGlu                          
104510501055                                                              
AspAlaProProValAsnGlnMetValLysValGluLeuLeuGluGlu                          
106010651070                                                              
ArgAsnThrAlaLeuGlyValIleSerAsnTrpThrAspGluLeuArg                          
107510801085                                                              
LeuProProCysThrIlePheGlnAlaPheLysTyrTyrLeuAspIle                          
109010951100                                                              
ThrThrProProThrProLeuGlnLeuGlnGlnPheAlaSerLeuAla                          
1105111011151120                                                          
ThrSerGluLysGluLysGlnArgLeuLeuValLeuSerLysGlyLeu                          
112511301135                                                              
GlnGluTyrGluGluTrpLysTrpGlyLysAsnProThrIleValGlu                          
114011451150                                                              
ValLeuGluGluPheProSerIleGlnMetProAlaThrLeuLeuLeu                          
115511601165                                                              
ThrGlnLeuSerLeuLeuGlnProArgTyrTyrSerIleSerSerSer                          
117011751180                                                              
ProAspMetTyrProAspGluValHisLeuThrValAlaIleValSer                          
1185119011951200                                                          
TyrArgThrArgAspGlyGluGlyProIleHisHisGlyValCysSer                          
120512101215                                                              
SerTrpLeuAsnArgIleGlnAlaAspGluLeuValProCysPheVal                          
122012251230                                                              
ArgGlyAlaProSerPheHisLeuProArgAsnProGlnValProCys                          
123512401245                                                              
IleLeuValGlyProGlyThrGlyIleAlaProPheArgSerPheTrp                          
125012551260                                                              
GlnGlnArgGlnPheAspIleGlnHisLysGlyMetAsnProCysPro                          
1265127012751280                                                          
MetValLeuValPheGlyCysArgGlnSerLysIleAspHisIleTyr                          
128512901295                                                              
ArgGluGluThrLeuGlnAlaLysAsnLysGlyValPheArgGluLeu                          
130013051310                                                              
TyrThrAlaTyrSerArgGluProAspLysProLysLysTyrValGln                          
131513201325                                                              
AspIleLeuGlnGluGlnLeuAlaGluSerValTyrArgAlaLeuLys                          
133013351340                                                              
GluGlnGlyGlyHisIleTyrValCysGlyAspValThrMetAlaAla                          
1345135013551360                                                          
AspValLeuLysAlaIleGlnArgIleMetThrGlnGlnGlyLysLeu                          
136513701375                                                              
SerAlaGluAspAlaGlyValPheIleSerArgMetArgAspAspAsn                          
138013851390                                                              
ArgTyrHisGluAspIlePheGlyValThrLeuArgThrIleGluVal                          
139514001405                                                              
ThrAsnArgLeuArgSerGluSerIleAlaPheIleGluGluSerLys                          
141014151420                                                              
LysAspThrAspGluValPheSerSer                                               
14251430                                                                  
(2) INFORMATION FOR SEQ ID NO:22:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 256 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: EPO-1 HRE element                                 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                  
GAACTGAAACCACCAATATGACTCTTGGCTTTTCTGTTTTCTGGGAACCTCCAAATCCCC60            
TGGCTCTGTCCCACTCCTGGCAGCAGTGCAGCAGGTCCAGGTCCGGGAAATGAGGGGTGG120           
AGGGGGCTGGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGACCTACCGGCC180           
TAGGCCACAAGCTCTGCCTACGCTGGTCAATAAGGTGTCTCCATTCAAGGCCTCACCGCA240           
GTAAGGCAGCTGCCAA256                                                       
(2) INFORMATION FOR SEQ ID NO:23:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 42 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: 42 bp EPO 3'hypoxia response                      
enhancer element (Madan, et al, PNAS 90:3928, 1993)                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                  
GGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACCTCTCGA42                              
(2) INFORMATION FOR SEQ ID NO:24:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 86 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: 86 nucleotide fragment from                       
αMHC promoter                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                  
GTCCCAGCAGATGACTCCAAATTTAGGCAGCAGGCACGTGGAATGAGCTATAAAGGGGCT60            
GGAGCGCTGAGAGCTGTCAGACCGAG86                                              
(2) INFORMATION FOR SEQ ID NO:25:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 2423 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: unknown                                                     
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: mouse catalase gene GenBank #L25069               
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 88..1671                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                  
ATTGCCTTCTCCGGGTGGAGACCAGACCGCTGCGTCCGTCCCTGCTGTCTCACGTTCCGC60            
AGCTCTGCAGCTCCGCAATCCTACACCATGTCGGACAGTCGGGACCCAGCC111                    
MetSerAspSerArgAspProAla                                                  
15                                                                        
AGCGACCAGATGAAGCAGTGGAAGGAGCAGCGGGCCTCGCAGAGACCT159                       
SerAspGlnMetLysGlnTrpLysGluGlnArgAlaSerGlnArgPro                          
101520                                                                    
GATGTCCTGACCACCGGAGGCGGGAACCCAATAGGAGATAAACTTAAT207                       
AspValLeuThrThrGlyGlyGlyAsnProIleGlyAspLysLeuAsn                          
25303540                                                                  
ATCATGACCGCGGGGTCCCGAGGGCCCCTCCTCGTTCAGGATGTGGTT255                       
IleMetThrAlaGlySerArgGlyProLeuLeuValGlnAspValVal                          
455055                                                                    
TTCACTGACGAGATGGCACACTTTGACAGAGAGCGGATTCCTGAGAGA303                       
PheThrAspGluMetAlaHisPheAspArgGluArgIleProGluArg                          
606570                                                                    
GTGGTACACGCAAAAGGAGCAGGTGCTTTTGGATACTTTGAGGTCACC351                       
ValValHisAlaLysGlyAlaGlyAlaPheGlyTyrPheGluValThr                          
758085                                                                    
CACGATATCACCAGATACTCCAAGGGAAAGGTGTTTGAGCATATTGGA399                       
HisAspIleThrArgTyrSerLysGlyLysValPheGluHisIleGly                          
9095100                                                                   
AAGAGGACCCCTATTGCCGTTCGGTTCTCCACAGTCGCTGGAGAGTCA447                       
LysArgThrProIleAlaValArgPheSerThrValAlaGlyGluSer                          
105110115120                                                              
GGCTCAGCTGACACAGTTCGTGACCCTCGGGGGTTTGCAGTGAAATTT495                       
GlySerAlaAspThrValArgAspProArgGlyPheAlaValLysPhe                          
125130135                                                                 
TACACTGAAGATGGTAACTGGGATCTTGTGGGAAACAACACCCCTATT543                       
TyrThrGluAspGlyAsnTrpAspLeuValGlyAsnAsnThrProIle                          
140145150                                                                 
TTCTTCATCAGGGATGCCATATTGTTTCCATCCTTTATCCATAGCCAG591                       
PhePheIleArgAspAlaIleLeuPheProSerPheIleHisSerGln                          
155160165                                                                 
AAGAGAAACCCACAGACTCACCTGAAGGATCCTGACATGGTCTGGGAC639                       
LysArgAsnProGlnThrHisLeuLysAspProAspMetValTrpAsp                          
170175180                                                                 
TTCTGGAGTCTTCGTCCCGAGTCTCTCCATCAGGTTTCTTTCTTGTTC687                       
PheTrpSerLeuArgProGluSerLeuHisGlnValSerPheLeuPhe                          
185190195200                                                              
AGTGACCGAGGGATTCCCGATGGTCACCGGCACATGAATGGCTATGGA735                       
SerAspArgGlyIleProAspGlyHisArgHisMetAsnGlyTyrGly                          
205210215                                                                 
TCACACACCTTCAAGTTGGTTAATGCAGATGGAGAGGCAGTCTATTGC783                       
SerHisThrPheLysLeuValAsnAlaAspGlyGluAlaValTyrCys                          
220225230                                                                 
AAGTTCCATTACAAGACCGACCAGGGCATCAAAAACTTGCCTGTTGGA831                       
LysPheHisTyrLysThrAspGlnGlyIleLysAsnLeuProValGly                          
235240245                                                                 
GAGGCAGGAAGGCTTGCTCAGGAAGATCCGGATTATGGCCTCCGAGAT879                       
GluAlaGlyArgLeuAlaGlnGluAspProAspTyrGlyLeuArgAsp                          
250255260                                                                 
CTTTTCAATGCCATCGCCAATGGCAATTACCCGTCCTGGACGTTTTAC927                       
LeuPheAsnAlaIleAlaAsnGlyAsnTyrProSerTrpThrPheTyr                          
265270275280                                                              
ATCCAGGTCATGACTTTTAAGGAGGCAGAAACTTTCCCATTTAATCCA975                       
IleGlnValMetThrPheLysGluAlaGluThrPheProPheAsnPro                          
285290295                                                                 
TTTGATCTGACCAAGGTTTGGCCTCACAAGGACTACCCTCTTATACCA1023                      
PheAspLeuThrLysValTrpProHisLysAspTyrProLeuIlePro                          
300305310                                                                 
GTTGGCAAAGTGGTTTTAAACAAAAATCCAGTTAATTACTTTGCTGAA1071                      
ValGlyLysValValLeuAsnLysAsnProValAsnTyrPheAlaGlu                          
315320325                                                                 
GTTGAACAGATGGCTTTTGACCCAAGCAATATGCCCCCTGGCATCGAG1119                      
ValGluGlnMetAlaPheAspProSerAsnMetProProGlyIleGlu                          
330335340                                                                 
CCCAGCCCTGACAAAAAGCTTCAGGGCCGCCTTTTTGCCTACCCGGAC1167                      
ProSerProAspLysLysLeuGlnGlyArgLeuPheAlaTyrProAsp                          
345350355360                                                              
ACTCACCGCCACCGCCTGGGACCCAACTATCTGCAGATACCTGTGAAC1215                      
ThrHisArgHisArgLeuGlyProAsnTyrLeuGlnIleProValAsn                          
365370375                                                                 
TGTCCCTACCGCGCTCGAGTGGCCAACTACCAGCGTGATGGCCCCATG1263                      
CysProTyrArgAlaArgValAlaAsnTyrGlnArgAspGlyProMet                          
380385390                                                                 
TGCATGCATGACAACCAGGGTGGTGCCCCCAACTATTACCCCAACAGC1311                      
CysMetHisAspAsnGlnGlyGlyAlaProAsnTyrTyrProAsnSer                          
395400405                                                                 
TTCAGCGCACCAGAGCAGCAGCGCTCAGCCCTGGAGCACAGCGTCCAG1359                      
PheSerAlaProGluGlnGlnArgSerAlaLeuGluHisSerValGln                          
410415420                                                                 
TGCGCTGTAGATGTGAAACGCTTCAACAGTGCTAATGAAGACAATGTC1407                      
CysAlaValAspValLysArgPheAsnSerAlaAsnGluAspAsnVal                          
425430435440                                                              
ACTCAGGTGCGGACATTCTACACAAAGGTGTTGAATGAGGAGGAGAGG1455                      
ThrGlnValArgThrPheTyrThrLysValLeuAsnGluGluGluArg                          
445450455                                                                 
AAACGCCTGTGTGAGAACATTGCCGGCCACCTGAAGGACGCTCAGCTT1503                      
LysArgLeuCysGluAsnIleAlaGlyHisLeuLysAspAlaGlnLeu                          
460465470                                                                 
TTCATTCAGAAGAAAGCGGTCAAGAATTTCACTGACGTCCACCCTGAC1551                      
PheIleGlnLysLysAlaValLysAsnPheThrAspValHisProAsp                          
475480485                                                                 
TATGGGGCCCGCATCCAGGCTCTTCTGGACAAGTACAACGCTGAGAAG1599                      
TyrGlyAlaArgIleGlnAlaLeuLeuAspLysTyrAsnAlaGluLys                          
490495500                                                                 
CCTAAGAACGCAATTCACACCTACACGCAGGCCGGCTCTCACATGGCT1647                      
ProLysAsnAlaIleHisThrTyrThrGlnAlaGlySerHisMetAla                          
505510515520                                                              
GCGAAGGGAAAAGCTAACCTGTAACTCCGGTGCTCAGCCTCCGCTGAGGAG1698                   
AlaLysGlyLysAlaAsnLeu                                                     
525                                                                       
ACCTCTCGTGAAGCCGAGCCTGAGGATCACCTGTAATCAACGCTGGATGGATTCTCCCCC1758          
GCCGGAGCGCAGACTCACGCTGATGACTTTAAAACGATAATCCGGGCTTCTAGAGTGAAT1818          
GATAACCATGCTTTTGATGCCGTTTCCTGAAGGGAAATGAAAGGTTAGGGCTTAGCAATC1878          
ATTTAACAGAAACATGGATCTAATAGGACTTCTGTTTGGATTATTCATTTAAATGACTAC1938          
ATTTAAAATGATTACAAGAAAGGTGTTCTAGCCAGAAACATGACTTGATTAGACAAGATA1998          
AAAATCTTGGCGAGAATAGTGTATTCTCCTATTACCTCATGGTCTGGTATATATACAATA2058          
CAACACACATACCACACACACACACACATGCAATACACACACTACACACACATACACACA2118          
CTCACACACACTCATACACACACATGAAGAGATGATAAAGATGGCCCACTCAGAATTTTT2178          
TTTTTATTTTTCTAAGGTCCTTATAAGCAAAACCATACTTGCATCATGTCTTCCAAAAGT2238          
AACTTTAGCACTGTTGAAACTTAATGTTTATTCCTGTGCTGTGCGGTGCTGTGCTGTGCT2298          
GTGCTGTGCAGCTAATCAGATTCTTGTTTTTTCCCACTTGGATTATGTTGATGCTAATAC2358          
GCAGTGATTTCACATAGGATGATTTGTACTTGCTTACATTTTTACAATAAAATGATCTAC2418          
ATGGA2423                                                                 
(2) INFORMATION FOR SEQ ID NO:26:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 527 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                  
MetSerAspSerArgAspProAlaSerAspGlnMetLysGlnTrpLys                          
151015                                                                    
GluGlnArgAlaSerGlnArgProAspValLeuThrThrGlyGlyGly                          
202530                                                                    
AsnProIleGlyAspLysLeuAsnIleMetThrAlaGlySerArgGly                          
354045                                                                    
ProLeuLeuValGlnAspValValPheThrAspGluMetAlaHisPhe                          
505560                                                                    
AspArgGluArgIleProGluArgValValHisAlaLysGlyAlaGly                          
65707580                                                                  
AlaPheGlyTyrPheGluValThrHisAspIleThrArgTyrSerLys                          
859095                                                                    
GlyLysValPheGluHisIleGlyLysArgThrProIleAlaValArg                          
100105110                                                                 
PheSerThrValAlaGlyGluSerGlySerAlaAspThrValArgAsp                          
115120125                                                                 
ProArgGlyPheAlaValLysPheTyrThrGluAspGlyAsnTrpAsp                          
130135140                                                                 
LeuValGlyAsnAsnThrProIlePhePheIleArgAspAlaIleLeu                          
145150155160                                                              
PheProSerPheIleHisSerGlnLysArgAsnProGlnThrHisLeu                          
165170175                                                                 
LysAspProAspMetValTrpAspPheTrpSerLeuArgProGluSer                          
180185190                                                                 
LeuHisGlnValSerPheLeuPheSerAspArgGlyIleProAspGly                          
195200205                                                                 
HisArgHisMetAsnGlyTyrGlySerHisThrPheLysLeuValAsn                          
210215220                                                                 
AlaAspGlyGluAlaValTyrCysLysPheHisTyrLysThrAspGln                          
225230235240                                                              
GlyIleLysAsnLeuProValGlyGluAlaGlyArgLeuAlaGlnGlu                          
245250255                                                                 
AspProAspTyrGlyLeuArgAspLeuPheAsnAlaIleAlaAsnGly                          
260265270                                                                 
AsnTyrProSerTrpThrPheTyrIleGlnValMetThrPheLysGlu                          
275280285                                                                 
AlaGluThrPheProPheAsnProPheAspLeuThrLysValTrpPro                          
290295300                                                                 
HisLysAspTyrProLeuIleProValGlyLysValValLeuAsnLys                          
305310315320                                                              
AsnProValAsnTyrPheAlaGluValGluGlnMetAlaPheAspPro                          
325330335                                                                 
SerAsnMetProProGlyIleGluProSerProAspLysLysLeuGln                          
340345350                                                                 
GlyArgLeuPheAlaTyrProAspThrHisArgHisArgLeuGlyPro                          
355360365                                                                 
AsnTyrLeuGlnIleProValAsnCysProTyrArgAlaArgValAla                          
370375380                                                                 
AsnTyrGlnArgAspGlyProMetCysMetHisAspAsnGlnGlyGly                          
385390395400                                                              
AlaProAsnTyrTyrProAsnSerPheSerAlaProGluGlnGlnArg                          
405410415                                                                 
SerAlaLeuGluHisSerValGlnCysAlaValAspValLysArgPhe                          
420425430                                                                 
AsnSerAlaAsnGluAspAsnValThrGlnValArgThrPheTyrThr                          
435440445                                                                 
LysValLeuAsnGluGluGluArgLysArgLeuCysGluAsnIleAla                          
450455460                                                                 
GlyHisLeuLysAspAlaGlnLeuPheIleGlnLysLysAlaValLys                          
465470475480                                                              
AsnPheThrAspValHisProAspTyrGlyAlaArgIleGlnAlaLeu                          
485490495                                                                 
LeuAspLysTyrAsnAlaGluLysProLysAsnAlaIleHisThrTyr                          
500505510                                                                 
ThrGlnAlaGlySerHisMetAlaAlaLysGlyLysAlaAsnLeu                             
515520525                                                                 
(2) INFORMATION FOR SEQ ID NO:27:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 969 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: unknown                                                     
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: human manganese superoxide dismutase              
EMBL #X59445                                                              
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 61..729                                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                  
TGGCTTCGGCAGCGGCTTCAGCAGATCGGCGGCATCAGCGGTAGCACCAGCACTAGCAGC60            
ATGTTGAGCCGGGCAGTGTGCGGCACCAGCAGGCAGCTGGCTCCGGCT108                       
MetLeuSerArgAlaValCysGlyThrSerArgGlnLeuAlaProAla                          
151015                                                                    
TTGGGGTATCTGGGCTCCAGGCAGAAGCACAGCCTCCCCGACCTGCCC156                       
LeuGlyTyrLeuGlySerArgGlnLysHisSerLeuProAspLeuPro                          
202530                                                                    
TACGACTACGGCGCCCTGGAACCTCACATCAACGCGCAGATCATGCAG204                       
TyrAspTyrGlyAlaLeuGluProHisIleAsnAlaGlnIleMetGln                          
354045                                                                    
CTGCACCACAGCAAGCACCACGCGGCCTACGTGAACAACCTGAACGTC252                       
LeuHisHisSerLysHisHisAlaAlaTyrValAsnAsnLeuAsnVal                          
505560                                                                    
AACGAGGAGAAGTACCAGGAGGCGTTGGCCAAGGGAGATGTTACAGCC300                       
AsnGluGluLysTyrGlnGluAlaLeuAlaLysGlyAspValThrAla                          
65707580                                                                  
CAGATAGCTCTTCAGCCTGCACTGAAGTTCAATGGTGGTGGTCATATC348                       
GlnIleAlaLeuGlnProAlaLeuLysPheAsnGlyGlyGlyHisIle                          
859095                                                                    
AATCATAGCATTTTCTGGACAAACCTCAGCCCTAACGGTGGTGGAGAA396                       
AsnHisSerIlePheTrpThrAsnLeuSerProAsnGlyGlyGlyGlu                          
100105110                                                                 
CCCAAAGGGGAGTTGCTGGAAGCCATCAAACGTGACTTTGGTTCCTTT444                       
ProLysGlyGluLeuLeuGluAlaIleLysArgAspPheGlySerPhe                          
115120125                                                                 
GACAAGTTTAAGGAGAAGCTGACGGCTGCATCTGTTGGTGTCCAAGGC492                       
AspLysPheLysGluLysLeuThrAlaAlaSerValGlyValGlnGly                          
130135140                                                                 
TCAGGTTGGGGTTGGCTTGGTTTCAATAAGGAACGGGGACACTTACAA540                       
SerGlyTrpGlyTrpLeuGlyPheAsnLysGluArgGlyHisLeuGln                          
145150155160                                                              
ATTGCTGCTTGTCCAAATCAGGATCCACTGCAAGGAACAACAGGCCTT588                       
IleAlaAlaCysProAsnGlnAspProLeuGlnGlyThrThrGlyLeu                          
165170175                                                                 
ATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCTTACTACCTTCAG636                       
IleProLeuLeuGlyIleAspValTrpGluHisAlaTyrTyrLeuGln                          
180185190                                                                 
TATAAAAATGTCAGGCCTGATTATCTAAAAGCTATTTGGAATGTAATC684                       
TyrLysAsnValArgProAspTyrLeuLysAlaIleTrpAsnValIle                          
195200205                                                                 
AACTGGGAGAATGTAACTGAAAGATACATGGCTTGCAAAAAGTAAACCACGA736                   
AsnTrpGluAsnValThrGluArgTyrMetAlaCysLysLys                                
210215220                                                                 
TCGTTATGCTGAGTATGTTAAGCTCTTTATGACTGTTTTTGTAGTGGTATAGAGTACTGC796           
AGAATACAGTAAGCTGCTCTATTGTAGCATTTCTTGATGTTGCTTAGTCACTTATTTCAT856           
AAACAACTTAATGTTCTGAATAATTTCTTACTAAACATTTTGTTATTGGGCAAGTGATTG916           
AAAATAGTAAATGCTTTGTGTGATTGAAAAAAAAAAAAAAAAAAAAAAAAAAA969                  
(2) INFORMATION FOR SEQ ID NO:28:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 222 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                  
MetLeuSerArgAlaValCysGlyThrSerArgGlnLeuAlaProAla                          
151015                                                                    
LeuGlyTyrLeuGlySerArgGlnLysHisSerLeuProAspLeuPro                          
202530                                                                    
TyrAspTyrGlyAlaLeuGluProHisIleAsnAlaGlnIleMetGln                          
354045                                                                    
LeuHisHisSerLysHisHisAlaAlaTyrValAsnAsnLeuAsnVal                          
505560                                                                    
AsnGluGluLysTyrGlnGluAlaLeuAlaLysGlyAspValThrAla                          
65707580                                                                  
GlnIleAlaLeuGlnProAlaLeuLysPheAsnGlyGlyGlyHisIle                          
859095                                                                    
AsnHisSerIlePheTrpThrAsnLeuSerProAsnGlyGlyGlyGlu                          
100105110                                                                 
ProLysGlyGluLeuLeuGluAlaIleLysArgAspPheGlySerPhe                          
115120125                                                                 
AspLysPheLysGluLysLeuThrAlaAlaSerValGlyValGlnGly                          
130135140                                                                 
SerGlyTrpGlyTrpLeuGlyPheAsnLysGluArgGlyHisLeuGln                          
145150155160                                                              
IleAlaAlaCysProAsnGlnAspProLeuGlnGlyThrThrGlyLeu                          
165170175                                                                 
IleProLeuLeuGlyIleAspValTrpGluHisAlaTyrTyrLeuGln                          
180185190                                                                 
TyrLysAsnValArgProAspTyrLeuLysAlaIleTrpAsnValIle                          
195200205                                                                 
AsnTrpGluAsnValThrGluArgTyrMetAlaCysLysLys                                
210215220                                                                 
(2) INFORMATION FOR SEQ ID NO:29:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 691 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: unknown                                                     
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: human enolase gene (EMBL #X56832)                 
fragment containinig nucleotides -628 to +63                              
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 629..691                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                  
CCTGGGGGTGGAGGTAGTAAAGGGTGAGCATGGTATTGGCTTGGAGGAAGTGGGGGACAT60            
TTCTGCTTTTTTTCCTCCTGGGACTGGAGATGCTTGAAAAAGCTGGGGGAAGGGGCGGCT120           
GGAGCAAGCAGATGGGACAAACTCTGGGAACACCGAAGGATCTAGGGAAAGGAGGCTGTG180           
AGGAGGGCAGCAGGGATGGATAGAAAAGGGCAGCTAGAGCTGGAACCTGATAGGGAATTG240           
GGGGCCCAAGGAGATTTCGGAGCAGGAAAATGAGAACCAGAAAGGATTTGAAGGCCACCA300           
GCCATGGAGAACAGACTGCTTGACCAGAGGGGTGGAAGGAGAAGGCCTAAGTGGAGGCTT360           
GGGGGAGGTGGGGGCTTGGTGAGCGGTGGCATCCCAGGAGCTATAGATAAGAGGCCCCTG420           
GATTCTTAGGATGGGAGGGTGGAATAAGAGCTGTTCTGAGTGGGGGAGGGGGCTGCGCCT480           
GCCTCTTTGGTCTGTGACCTTTTTGTAGGGTATTTTTAGCTCCAGCACCTGCCTTCTTGG540           
AGTGGGGAAGAATCTTAAAGGGCAAGGGATTTCTGGTTCCTTAAGAGATCAACTGTCTAC600           
ACTCACTCACACCTCCTGTCCTGCAGCCATGGCCATGCAGAAAATCTTTGCC652                   
MetAlaMetGlnLysIlePheAla                                                  
15                                                                        
CGGGAAATCTTGGACTCCAGGGGCAACCCCACGGTGGAG691                                
ArgGluIleLeuAspSerArgGlyAsnProThrValGlu                                   
101520                                                                    
(2) INFORMATION FOR SEQ ID NO:30:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 21 amino acids                                                
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                  
MetAlaMetGlnLysIlePheAlaArgGluIleLeuAspSerArgGly                          
151015                                                                    
AsnProThrValGlu                                                           
20                                                                        
(2) INFORMATION FOR SEQ ID NO:31:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 17 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(C) INDIVIDUAL ISOLATE: PKM/ENO3 consensus sequence                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                  
GAGAGGCGGGCTNNCTG17                                                       
__________________________________________________________________________

Claims (18)

It is claimed:
1. A chimeric DNA construct, comprising
a hypoxia response enhancer element, a tissue-specific promoter heterologous to the element, and a coding sequence, with the proviso that the coding sequence is not an alpha globin coding sequence,
wherein said promoter is operably linked to said coding sequence and said element is effective to cause expression of said coding sequence under conditions of hypoxia.
2. A chimeric DNA construct of claim 1, wherein said promoter is a cardiac-specific promoter.
3. A chimeric DNA construct of claim 1, wherein said promoter is selected from the group consisting of α-MHC5.5 promoter, α-MHC87 promoter, and human cardiac actin promoter.
4. A chimeric DNA construct of claim 1, wherein said promoter is a kidney-specific promoter.
5. A chimeric DNA construct of claim 4, wherein said promoter is a renin promoter.
6. A chimeric DNA construct of claim 1, wherein said promoter is a brain-specific promoter.
7. A chimeric DNA construct of claim 6, wherein said promoter is selected from the group consisting of aldolase C promoter, and tyrosine hydroxylase promoter.
8. A chimeric DNA construct of claim 1, wherein said promoter is a vascular endothelium-specific promoter.
9. A chimeric DNA construct of claim 8, wherein said promoter is selected from the group consisting of Et-1 promoter and vonWillebrand factor promoter.
10. A chimeric DNA construct of claim 1, wherein said hypoxia response enhancer element is selected from the group consisting of erythropoietin HRE element (HREE1), pyruvate kinase (PKM) HRE element, enolase 3 (ENO3) HRE element and endothelin-1 (ET-1) HRE element.
11. A chimeric DNA construct of claim 1, wherein said coding sequence is selected from the group consisting of nitric oxide synthase (NOS), Bcl-2, superoxide dismutase (SOD), and catalase.
12. An expression vector, comprising
a hypoxia response enhancer element, a tissue-specific promoter heterologous to the element, and a coding sequence, with the proviso that the coding sequence is not an alpha globin coding sequence
wherein said promoter is operably linked to said coding sequence and said element is effective to cause expression of said coding sequence.
13. An expression vector of claim 12, wherein said expression vector is a plasmid.
14. An expression vector of claim 12, wherein said expression vector is an adenovirus vector.
15. An expression vector of claim 12, wherein said expression vector is a retrovirus vector.
16. The chimeric DNA construct of claim 1, wherein said coding sequence is a viral thymidine kinase coding sequence.
17. The chimeric DNA construct of claim 16, wherein said viral thymidine kinase coding sequence encodes herpes simplex viral thymidine kinase.
18. The chimeric DNA construct of claim 1, wherein said coding sequence encodes luciferase.
US08/365,486 1994-12-23 1994-12-23 Tissue specific hypoxia regulated therapeutic constructs Expired - Fee Related US5834306A (en)

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PCT/IB1995/000996 WO1996020276A1 (en) 1994-12-23 1995-11-13 Tissue specific hypoxia regulated therapeutic constructs
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CA002208599A CA2208599A1 (en) 1994-12-23 1995-11-13 Tissue specific hypoxia regulated therapeutic constructs
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